Xylanase production

ABSTRACT

An isolated DNA with a nucleotide sequence encoding a ripening form of a xylanase fungal origin having bread improving activity. Cells are transformed with this DNA and used to produce the ripening form of xylanase which itself is suitable for use in flour and dough.

This invention lies in the field of recombinant DNA technology and isdirected at a cell having a certain function in a process, containingrecombinant DNA encoding at least one enzyme. The invention is directedespecially at a cell having a function in the field of food processingand also at cells with a function in processes in which acellulose-containing raw material is used, such as processes forpreparing beer, paper, starch, gluten etc, and processes for decomposingcellulose-containing waste such as agricultural waste, waste from paperrills etc.

In particular the invention is directed at cells having a function inthe process of fermentation, more especially at cells with a function inthe process of preparing bakery products.

The cell according to the invention is characterized in that the cellbecomes polyfunctional for the process in which it has a function, uponexpression of the recombinant DNA encoding at least one enzyme. In thecase of a fermentation process for example, such as the preparation ofbread, yeast is used as a cell with a particular function in saidprocess. A yeast cell according to the invention does not only have itsnormal function, i.c. a function that a yeast lacking the recombinantDNA can also carry out, but also has another function in said process ofbread preparation. An example of such an additional function is theexpression and secretion of a bread improving enzyme.

The present invention is directed in particular at a cell with afunction in the preparation of bakery products. Cells containingrecombinant DNA encoding enzymes selected from the group of enzymes withamylolytic and/or hemicellulolytic and/or cellulolytic activity aresuitable.

The invention is also directed at a process for the production of atleast one enzyme by a polyfunctional cell as described above comprisingculturing such a polyfunctional cell in a suitable nutrient medium andoptionally isolating the resulting enzyme form. In such a process, saidenzyme is preferably selected from the group of enzymes with amylolyticand/or hemicellulolytic and/or cellulolytic activity. A suitable mediumfor carrying out the process according to the invention can consist ofthe medium in which the process for which the cell is polyfunctional iscarried out. In the process of the preparation of a bakery product forexample said medium can be the dough that it to be baked. Naturally theother usual media for culturing cells can also be used. The choice ofmedia will depend on whether the enzyme is to be used in situ or has tobe isolated. In some cases it will suffice to use the medium containingsaid enzyme and in other cases the enzyme will have to be isolated fromthe medium.

The invention is also directed at an enzyme encoded by the recombinantDNA in said polyfunctional cell, whereby said enzyme is obtainable fromsuch a polyfunctional cell via the afore mentioned process for producingan enzyme. The invention is further directed at the use of such apolyfunctional cell or such an enzyme, for example in the processesdescribed above, such as food processing and processes using a cellulosecontaining raw material, preferably in a process for the preparation ofa bakery product.

Flour, yeast, water and salt are the basic ingredients of bread andother bakery products. For centuries materials having a positive effecton the manageability of the dough or the quality of the baked producthave been added in the manufacture of bread and similar bakery products,for the sake of convenience further referred to as bread making. Saidadditives, referred to as “bread improvers”, contain enzymes from maltor of microbial origin which play an important part in the differentphases of bread making, namely, the preparation of the bread batter,fermentation, baking, and storage of the bread product.

One of the relevant characteristics of bread that is influenced byadding specific enzymes is the so-called bread volume. In order toobtain a high bread volume in practice compositions containingcellulolytic, hemicellulolytic and/or amylolytic enzymes are added. Thecommercially available compositions of microbial origin, mostlyoriginating from a fungus o; one of the genera Aspergillus andTrichoderma, are substantially unpurified complex mixtures of differentenzyme activities, whereby it is not exactly known which enzymes arepresent in the composition and which have a bread improving activity.This lack of knowledge impedes further bread improvement and especiallyimpedes the control of the different dough processing and breadproperties, such as the bread volume.

Further investigation into the process of preparation of bakery productsresulted in the discovery that, in addition to α-amylase, at least axylanase enzyme is also of importance for the bread volume. A xylanaseis an enzyme that catalyzes the degradation of xylans occurring in thepentosan part of starch “tailings”. The term “tailings” is directed at afraction of, e.g., wheat starch consisting of water-insolublehemicellulose (pentosans and arabinoxylans) and damaged starch. Thisfraction is formed as the intermediate or top layer of the starch pelletduring centrifugation of a dough suspension obtained by washing dough toremove the gluten fraction.

Different xylanases have already been described in the literature,including xylanases of the bacterial species Bacillus pumilus(Panbangred et al., Mol. Gen. Genet. 192, 335-341, 1983, and Fukusaki etal., FEBS Lett. 321, 197-201, 1984), Bacillus subtilis (Paice et al.,Arch. Microbiol. 144, 201-206, 1986), and Bacillus circulans (Yang etal., Nucl. Acids Res. 16, 7187, 1988), of the yeast Aureobasidium(Leathers, Biotech. Lett. 12, 775-780, 1988) and of the fungusAspergillus niger (Fournier et al., Biotechnology and Bioengineering 27,539-546, 1985).

It is known from European patent application EP-A-0338452 that theproperties of dough and the quality of bread can be improved by addingdifferent enzyme compositions to the dough, including an enzymecomposition having hemicellulose degrading or xylanase activity, theorigin of which is not further specified. Such a hemicellulolytic enzymecomposition is a relatively undefined enzyme mixture which may containdifferent hemicellulolytic enzymes having various effects on the doughand bread properties. The presence of xylanases having bread improvingactivity to a smaller or greater extent is the coincidental result ofthe manner in which the enzyme composition that is intended as a breadimprover has been obtained. A controlled further optimization of breadimprovers, however, was not possible due to the lack of the requiredknowledge and of suitable recombinant DNA constructs encoding a xylanasehaving broad improving activity that could be used for a high productionof such a xylanase.

For the purpose of this invention, “bread improving activity” isgenerally taken to mean a favourable effect on any property of theprepared bakery product (including bread) or of the dough from which thebakery or bread product is made, and is particularly taken to mean afavourable effect on the bread volume.

The investigation on which the invention is based has extended to theidentification and cloning of a gene (xylA) encoding an enzyme, xylanasehaving bread improving activity, originating from a fungus of thespecies Aspergillus niger var. awamori, as well as to the transformationof different species of host cells in such a manner that the gene isexpressed or can be expressed in said host cells. The invention,however, comprises all xylanase genes originating from fungi andespecially from fungal strains from the same genus and therefore theinvention is not limited to the actually cloned gene.

The present invention is therefore also directed at recombinant DNAmaterial comprising DNA with a nucleotide sequence encoding at least aripening form of a xylanase of fungal origin.

The term “ripening form” refers to the different forms in which theenzyme may occur after expression of the associated gene. More inparticular, it refers to both the naturally and the not naturallyoccurring prepro-, pre- and pro-forms and to the ultimate mature form ofthe enzyme resulting after cleavage of a “leader” peptide.

More in particular, the invention, relates to recombinant DNA materialcomprising DNA with a nucleotide sequence encoding at least a ripeningform of a xylanase of Aspergillus origin.

Preferably, this aspect of the invention is concerned with recombinantDNA material comprising DNA with a nucleotide sequence encoding aripening form of a xylanase of Aspergillus niger origin, especially ofAspergillus niger var. awamori origin.

A preferred embodiment of this aspect of the invention is recombinantDNA material comprising DNA with a nucleotide sequence encoding at leasta ripening form of xylanase with an amino acid sequence as shown in FIG.1 (SEQ ID NO:7), and more in particular recombinant DNA materialcomprising DNA with a nucleotide sequence encoding a ripening form ofxylanase, as shown in FIG. 1 (SEQ ID NO:7). The invention is alsodirected at recombinant DNA material comprising DNA with a nucleotidesequence encoding at least a ripening form of xylanase with a nucleotidesequence that is equivalent to the nucleotide sequence of FIG. 1 (SEQ IDNO:7) with deletions, insertions or alterations in comparison to thenucleotide sequence of FIG. 1 (SEQ ID NO:7) such that the nucleotidesequence with deletions, insertions or alterations corresponds either tothe amino acid sequence as shown in FIG. 1 (SEQ ID NO:7) or to thoseparts of the amino acid sequence of FIG. 1 (SEQ ID NO:7) essential foran active ripening form of xylanase such as mature xylanase or activepre(pro) xylanase or the nucleotide sequence with deletions, insertionsor alterations has a complementary strand capable of hybridizing underhybridizing conditions to the nucleotide sequence of FIG. 1 (SEQ IDNO:7).

The the recombinant DNA according to the invention contains at least asequence encoding the fungal, in particular the Aspergillus xylanaseripening form. In addition, the recombinant DNA may contain many othertypes of information, such as regulating sequences (especially atranscription promoter) and a vector part usually provided with one ormore marker genes. These other types of information will often beconnect d with the selected host. Thus, for instance, the vector, themarker genes and the regulating sequences will be selected depending onthe selected host.

The recombinant DNA encoding at least a ripening xylanase of fungalorigin, however, may also contain other genes to be expressed in theselected host. Such a gene may advantageously encode at least one otherenzyme, wherein said other enzyme has amylolytic and/or hemicellulolyticand/or cellulolytic activity.

Another aspect of the invention is a cell containing genetic materialderived from recombinant DNA material according to the invention asdefined above, and more in particular such a cell capable of expressionof at least the xylanase ripening form encoded on said recombinant DNAmaterial. A preference exists for such a cell that is also apolyfunctional cell according to the invention and more especially forsuch a polyfunctional cell capable of expressing the recombinant DNAmaterial encoding a ripening form of xylanase of fungal origin underconditions present in raw material during preparation of a bakeryproduct.

Both a polyfunctional cell containing recombinant DNA encoding at leastone enzyme according to the invention, and a cell containing recombinantDNA material encoding a ripening form of xylanase of fungal originaccording to the invention (as well as the combination thereof) mayeither be a cell which is itself the direct result of gene manipulationor be a cell originating in any manner from a cell that has beentransformed by such gene manipulation. The invention further extends toboth live cells and cells that are no longer alive.

In principle the invention knows no special limitations with respect tothe nature of the cells, whereby those cells capable of expression of axylanase ripening form of fungal origin are preferred. However the cellsare preferably selected from the group consisting of bacterial cells,fungal cells, yeast cells, and plant cells.

Preferred examples of eminently suited host cells are

(a) fungal cells of one of the genera Aspergillus and Trichoderma, inparticular fungal cells of one of the species Aspergillus niger var.niger, Aspergillus niger var. awamori, Aspergillus nidulans, Aspergillusoryzae Trichoderma reisei and Trichoderma viride;

(b) yeast cells of one of the genera Saccharomyces, Kluyveromyces,Hansenula and Pichia, in particular yeast cells of one of the speciesSaccharomyces cerevisiae, Saccharomyces carlbergensis, Kluyveromyceslactis, Kluyveromyces marxianus, Hansenula polymorpha and Pichia Istoris;

(c) plant cells of a plant genus selected from the group consisting ofwheat, barley, oats, maize, pea, potato and tobacco, such as plant cellsof one of the species Solanum tuberosum and Nicotiana tabacum; and

(d) bacterial cells of one of the bacterial genera Bacillus,Lactobacillus and Streptococcus, such as bacteria of the speciesBacillus subtilis.

Cells according to the invention as defined above (polyfunctional and/orsimply containing recombinant DNA encoding a ripening form of xylanaseof fungal origin) may be important as agents for multiplying therecombinant DNA or as agents for producing at least one enzyme encodedon said recombinant DNA, such as the ripening form of xylanase.

In the case of enzyme production it is possible to use the cell toproduce enzyme and either isolate the enzyme from the culturing mediumor use the medium containing the enzyme after removal of the cells assuch, or in the case of the polyfunctional cells to use the cellsthemselves to produce the enzyme in situ in the process for which theyare poly-functional.

A direct use of the cells themselves is possible, e.g., if the hoststrain can be admitted without objection, in the production offoodstuffs as is the case for various fungal, yeast, plant, andbacterial species. In connection with bread making the yeast strainsthat are genetically manipulated in accordance with the presentinvention can for example be used directly.

Partly depending on the selected host the gene encoding xylanase will beused, either with or without introns occurring in said gene, either withits own transcription termination signals or originating from anothergene, and either with its own leader sequence or with a signal sequenceoriginating from another gene. For transformation of yeast, such asSaccharomyces cerevisiae (baker's yeast), it is preferable that theintrons are removed and that the own leader sequence is replaced by asignal sequence suitable for yeast, such as the signal sequence of theinvertase gene, ensuring correct processing and secretion of the matureprotein.

The removal of introns is necessary upon transformation of bacteria,such as Bacillus subtilis. In this case e.g. the α-amylase signalsequence can be used as signal sequence.

Suitable transformation methods and suitable expression vectors providedwith, e.g., a suitable transcription promoter, suitable transcriptiontermination signals, and suitable marker genes for selecting transformedcells are already known for many organisms, including differentbacterial, yeast, fungal, and plant species. Reference may be made foryeast for example to Tajima et al., Yeast 1, 67-77, 1985, which showsexpression of a foreign gene under control of the GAL7 promoterinducible by galactose in yeast, and for Bacillus subtilis for exampleto EP-A-0 157 441, describing a plasmid pMS48 containing the SPO2promoter as an expression vector. For other possibilities in these andother organisms reference is made to the general literature.

In another aspect the present invention consists of a ripening form of axylanase of a fungus, in particular of Aspergillus origin, obtained byexpression of recombinant DNA material according to the invention, asdefined above. Herein, special preference is given to a mature xylanasewith an amino acid sequence as illustrated in FIG. 1, as well as to apre(pro)-xylanase with an amino acid sequence as illustrated in FIG. 1(SEQ ID Nos: 7 and 8) and to any amino acid sequence of an activeequivalent form of xylanase comprising the amino acids of the sequenceof FIG. 1 which are essential for xylanase activity. The invention istherefore directed at an amino acid structure leading to a tertiaryenzyme structure with the same enzyme activity as the enzyme with thesequence of FIG. 1.

Yet another aspect of the invention consists of a process for producinga ripening form of a xylanase of a fungus, in particular of Aspergillusorigin, comprising culturing a polyfunctional cell capable of expressinga xylanase ripening form and or a cell capable of expressing therecombinant DNA material according to the invention encoding a ripeningform of xylanase of fungal origin in a suitable nutrient medium, andoptionally isolating the resulting xylanase ripening form. The term“isolating the resulting xylanase ripening form” also comprises apartial purification in which an enzyme composition is recoveredcomprising the relevant xylanase.

Further aspects of the present invention are a bread improvercomposition comprising an enzyme selected from the group of enzymes withamylolytic and/or hemicellulolytic and/or cellulolytic activity such asa ripening form of xylanase, in particular a mature xylanase of afungus, especially of Aspergillus origin, whereby said enzyme isobtainable from a polyfunctional cell according to the invention and/orfrom expression of recombinant DNA according to the invention encoding aripening form of a xylanase of fungal origin and a bread improvercomposition comprising a polyfunctional cell according to the invention;a flour and dough composition comprising an enzyme selected from thegroup of enzymes with amylolytic and/or hemicellulolytic and/orcellulolytic activity such as a ripening form of xylanase, in particulara mature xylanase of a fungus, especially of Aspergillus origin, wherebysaid enzyme is obtainable from a polyfunctional cell according to theinvention and/or from expression of recombinant DNA according to theinvention encoding a ripening form of a xylanase of fungal origin; aflour and dough composition comprising a polyfunctional cell accordingto the invention; a bakery product obtained using such flour or doughcompositions as described above; and a process for the preparation of abakery product, using such flour or dough compositions especially inwhich a mature xylanase of a fungus, in particular of Aspergillus originis included.

The invention, however, also extends to other uses of fungal xylanases,such as use within the scope of beer making, particularly thepreparation of beers on the basis of wheat, in order to improvefilterability, use in the paper-making industry to reduce waterabsorption by the paper material, use in the treatment of agriculturalwaste, etc.

The invention will now be elucidated by means of an extensivedescription of the identification, cloning and expression of a xylanasesuitable as a bread improver. In the experimental work described in theexamples the fungal strain Aspergillus niger var. awamori CBS 115.52(ATCC 11358) is used as a source for the xylanase. According toinvestigations carried out by the inventors, said strain, afterinduction with wheat bran, is capable of producing a xylanase havingbread improving properties, while the culture medium exhibits anα-amylase activity, a low glucanase activity and a low protease activityunder these induction conditions. The amount of xylanase produced by thewild-type strain, however, is too low for use in a commercial process.For this reason the invention also provides gene manipulations enablinga biotechnological production of the xylanase on a commercial scale.

The conducted experimental work comprises the isolation of the geneencoding a xylanase enzyme (the xylA gene) from a gene library ofchromosomal Aspergillus niger var. awamori DNA made in a λ vector. Forsaid isolation a probe was made with a composition derived from theN-terminal amino acid sequence of the purified mature protein asdetermined by the inventors. By means of this probe a number of λ cloneswere isolated which possibly contained the gene. A DNA fragment fromthese positive λ clones was subcloned. Subsequently, the DNA sequence ofpart of the cloned chromosomal DNA fragment was determined. By means ofthese results and those of mRNA analysis, the length of the xylA gene,the length of the mRNA, and the presence and position of an intron havebeen determined. It could be derived from the data that the xylA geneencodes a protein of 211 amino acids (a pre(pro)-form) in which themature protein of 184 amino acids is preceded by a “leader” peptide of27 residues.

Three expression vectors containing the xylanase gene including the xylAterminator have been constructed. In one of these vectors the xylA geneis preceded by its own expression signals. In the second vector the xylAexpression signals (up to the ATG codon) have been replaced by theconstitutive expression signals of the Aspergillus nidulansglyceraldehyde 3-phosphate dehydrogenase (gpdA) gene (see Punt et al.,Gene 69, 49-57, 1988), while in the third vector the xylA gene ispreceded by the inducible expression signals of the Aspergillus nigervar. niger glucoamylase (glaA) gene. All the expression vectors containthe Aspergillus nidulans acetamidase (amdS) gene as selection marker asdescribed by K. Wernars, “DNA mediated transformation of the filamentousfungus Aspergillus nidulans”, thesis, Landbouw Hogeschool Wageningen1986. By means of this selection marker transformants can be obtained inwhich the vector, and consequently also the xylA gene, is integratedinto the genome in a large number of copies.

Multicopy transformants were obtained by transformation of theAspergillus strains A. niger var. awamori and A. niger var. niger N402with the above mentioned expression vectors. In shaking flaskexperiments the production of xylanase was measured after culturing theresulting transformants in different media. The results (maximumproduction levels) are listed in Table A given below, in which thexylanase activity is expressed in 10³ units (U) per ml. A unit isdefined as the amount of enzyme which, per 1 minute, releases an amountof reducing groups from xylan equivalent to 1 mg xylose.

Survey of the maximum xylanase production levels in shaking flaskexperiments after culturing in different media strain promoter xylanrich medium starchbran A. niger xyl A s.c. 15 0 0 14 var. awamori A.niger xyl A s.c.  5 n.d. n.d. var. niger N402 A. niger xyl A m.c. 59 78var. awamori A. niger xyl A m.c. n.d. 120 var. niger N402 A. niger xyl Am.c. 36 140 var. niger AB4.1 A. niger gpdA m.c. 20 32 var. awamori A.niger gpdA m.c. 11 12 var. niger N402 A. niger glaA m.c. 71 45 var.awamori A. niger glaA m.c. 54 72 var. niger N402 s.c.: single copy wildtype strain m.c.: multicopy transformants n.d.: not determined

After induction with xylan the A. niger var. awamori and A. niger var.niger N402 “xylA” multicopy transformants with xylA promoter producemuch more xylanase than the wild type A. niger var. awamori and A. nigervar. niger strains. From this and from data obtained in the molecularanalysis of the gene it can be derived that the cloned gene encodes afunctional xylanase. Furthermore it is apparent from the afore mentionedthat the multicopy transformants are capable of overproduction of theactive enzyme. In baking tests this enzyme composition also has thedesired properties.

Multicopy transformants of the host strains with the heterologous gpdAor glaA promoter are also capable of an increased production of activexylanase. In rich medium the “gpdA” transformants produce a clearlylarger amount of xylanase than the wild type A. niger var. awamoristrain. However, the production levels observed in the conducted testsare substantially lower than the level obtained in the tests with “xylA”multicopy transformants. After induction with starch the productionlevels of “glaA” multicopy transformants are comparable to those of“xylA” multicopy transformants in xylan medium.

In medium with wheat bran the best A. niger var. awamori “xylA”multicopy transformants produce much more xylanase than is the case inxylan medium. In this medium the best A. niger var. niger N402 “xylA”transformants reach a very high xylanase production level. The highestproducing “gpdA” multicopy transformants of both A. niger var. awamoriand of A. niger var. niger N402 in bran produce as much xylanase as inrich medium. In medium with wheat bran the production by A. niger var.awamori “glaA” transformants is lower than in starch. In this medium,however, A. niger var. niger N402 “glaA” transformants produce more thanin starch.

The production reached by Aspergillus niger var. niger N402transformants is higher than that of Aspergillus niger var. awamoritransformants. The production level of the A. niger var. awamoritransformants, however, can be further increased by using suitable A.niger var. awamori mutant strains, such as A. niger var. awamori #40,which produces clearly more xylanase than the wild type strain. Themutant A. niger var. awamori #40 has been obtained by mutagenesis of A.niger var. awamori spores and selection for xylanase production. In branmedium the “xylA” A. niger var. awamori #40 transformant produced 190000 U xylanase, which is a considerable increase over the best producingA. niger var. awamori transformant.

Further experiments relate to the isolation and use of the thus producedxylanase as a bread improver (see example II) and expression experimentsin a yeast strain and a bacterium (examples III and IV, respectively).While example V demonstrates the use of a polyfunctional yeast accordingto the invention in the preparation of bread, whereby said yeastproduces xylanase during fermentation of lean bread dough.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO:7) shows the DNA sequence of a part of a ca 2.1 kbPtI-PstI Aspergillus niger var. awamori fragment present in the plasmidpAW14B, which fragment contains a gene encoding a xylanase, indicated asthe xylA gene. The translation start and the stop codon are doublyunderlined; The 49 bp intron is underlined. The start of the matureprotein is indicated. The amino acid acid sequence of the protein (bothof the pre(pro-form and of the mature protein) is also mentioned in FIG.1 (SEQ ID NO: 7), using the one-letter code.

FIG. 2 shows the restriction map of the genomic DNA region of A. nigervar. awamori, comprising the xylA gene cloned in the phages λ-1 andλ-14. The used abbreviations stand for: S: SalI; E: EcoRI; H: HindIII;P: PstI; B: BamHI; S#: SalI site originating from the polylinker ofλ-EMBL3; D: Sau3A. The massive bar indicates a 1.2 kb PstI*-BamHIfragment hybridizing with Xy106.

FIG. 3 shows the plasmid pAW14B obtained by an insertion of a 5.3 kb A.niger var. awamori SalI fragment in pUC19.

FIG. 4 shows the plasmid pAW14S containing the xylA gene with its ownpromoter and amds as a selection marker.

FIG. 5 shows the plasmid pAW14B-2 containing a translation fusion of thexylA gene with the A. nidulans gpdA promoter.

FIG. 6 shows the plasmid pAW14S-2 containing a translation fusion of thexylA gene with the Aspergillus nidulans gpdA promoter and amdS as aselection marker.

FIG. 7 shows the plasmid pAW14S-3 containing a translation fusion of thexylA gene with the Aspergillus niger glaA promoter and amds as aselection marker.

FIG. 8 (SEQ ID NO:s 9 and 33-43) shows the nucleotide sequences of theDNA fragment BAK1 and of the synthetic oligonucleotides from which thisfragment is built up.

FIG. 9 (SEQ ID NO:s 10 and 13-27) is a schemaic representation of theconstruction of the plasmid pBAK1.

FIG. 10 shows the nucleotide sequences of the DNA fragment BAK2 and ofthe synthetic oligonucleotides from which this fragment is built up.

FIGS. 11A and 11B are a schematic representation of the construction ofthe plasmid pBAK21.

FIGS. 12A and 12B are a schematic representation of the construction ofthe plasmid pUR2901.

FIGS. 13A and 13B are a schematic representation of the construction ofthe plasmid pUR2904.

FIGS. 14A and 14B are a schematic representation of the construction ofthe plasmid pDUR2921.

FIG. 15 (SEQ ID NO:s11, 27,19,20,28,25 and 29) shows the nucleotidesequences of the DNA fragment BAK4 and of the synthetic oligonucleotidesfrom which this fragment is built up.

FIGS. 16A and 16B is a schematic representation of the construction ofthe plasmid pUR2950.

FIGS. 17A and 17B are a schematic representation of the construction ofthe plasmid pUR2951.

FIG. 18 shows the nucleotide sequence of the in vitro amplified S.cerevisiae PGK promoter. In the double stranded sequence the primers areshown bold, the ATG start codon is on a shaded background, and therestriction sites EcoRI, BqlII, BspMI and HindIII are indicated.

FIG. 19 is a schematic representation of the construction of the plasmidpUR2918.

FIG. 20 (SEQ ID NO:s 15,30,18,19,31,22,23,24,25 and 32) shows thenucleotide sequences of the DNA fragment BAK5 and of the syntheticoligonucleotides from which this fragment is built up.

FIGS. 21A and 21B are a schematic representation of the construction ofthe plasmid pUR2920.

FIGS. 22A and 22B are a schematic representation of the construction ofthe plasmid pUR2922.

FIGS. 23A and 23B are a schematic representation of the construction ofthe plasmid pUR2923.

EXAMPLE I

Cloning and Characterisation of the Xylanase Gene (xylA) of Aspergillusniger var. awamori

1.1 Isolation of the Aspergillus niger var. awamori xylA Gene

In order to isolate the xylA gene from chromosomal DNA of Aspergillusniger var. awamori different probes were synthesized consisting ofmixtures of oligonucleotides (Table B). The composition of thesemixtures was derived from the N-terminal amino acid sequence of purifiedxylanase protein.

TABLE B Probes derived from the N-terminal amino acid sequence ofxylanase protein N-terminal amino acid sequence of xylanase protein:(SEQ ID NO:1) 1        5      10             15SerAlaGlyIleAsnTyrValGlnAsnTyrAsnGlyAsnLeuGlyAspPheprobe                 base sequence 3′-5′ Xy101 (SEQ IDNO:2)  TTAATACAXGTTTTAATATTACC                       G  G     C  G  G  G Xy104 (SEQ IDNO:3)           C G G C C G T A G T T G A T G C A G -GTCTTGATGTTGCCGTTGGACCCGCTGAA Xy105 (SEQ ID NO:4)ATGTTGCCATTAAAXCCACTGAA                           G   GG    G                          C   C Xy106 (SEQ ID NO:5)CGGCCGTAGTTGATGCAGGTCTTGATGTTGCCGTTGGAGCCGCTGAA                      C  C  C        C  T      C    C  C X = A, G, C orT Xy101:(SEQ ID NO:2) a mixture of 256 oligonucleotides having a lengthof 23 desoxynucleotides of which the sequence is complementary to thepart of the coding strand encoding the amino acids 5-12.

Xyl04(SEQ ID NO:3): an oligonucleotide having a length of 47desoxynucleotides of which the sequence is complementary to the part ofthe coding strand encoding the amino acids 2-17.

Xyl03(SEQ ID NO: 4) a mixture of 144 oligonucleotides having a length of23 desoxynucleotides of which the sequence is complementary to the partof the coding strand encoding the amino acids 10-17.

Xyl06(SEQ ID NO:5) a mixture of 256 oligonucleotides having a length of47 desoxynucleotides the sequence of which is complementary to the partof the coding strand encoding the amino acids 2-17.

In Xyl05(SEQ ID NO:4) and Xyl06(SEQ ID NO:5) not all of the bases thatcan possibly occur are introduced at the third position of the codons inorder to obtain no more than 256 oligonucleotides in the mixture.

By means of Southern blot analysis it was established that in digests ofchromosomal DNA—under stringent conditions—only one band hybridizes withthe probes used. In the EcoRI, SalT and BamHI digest of Aspergillusniger var. awamori DNA one band of respectively 4.4, 5.3 and 9.5 kbhybridizes with both Xyl01, Xyl04 and Xyl06(SEQ ID NO:s 2, SEQ ID NO:3and SEQ ID NO:5, respectively). With Xyl05(SEQ ID NO:4) no clear signalwas found at 41° C. On the basis of this result a λ gene library ofAspergillus niger var. awamori DNA was hybridized at 65° C. with theoligonucleotide mixture xyl06(SEQ ID NO:8) as a probe. Of the 65000tested plaques (corresponding to 32 times the genome) three plaques(λ-1, λ-14 and λ-63) hybridized with this probe. After hybridization ofdigests of λ-1 and λ-14 DNA with Xyl06 a hybridizing band of >10 kb wasfound in the EcoRI digest of λ-1. The size of the hybridizing band inthe λ-14 and the chromosomal EcoRI digest was 4.4. kb. In the SalIdigest of λ-1 a 4.6 kb band hybridizes; in the SalI digest of λ-14 thisis, like in chromosomal DNA, a 5.3 kb band. Also a 1.2 kp PstI-BamHIfragment (FIG. 2) hybridizes with Xyl06(SEQ ID NO:5). On the basis ofrestriction patterns with different enzymes and cross-hybridization ofλ-1 and λ-14 digests with the 5.3 kb SalI fragment of λ-14 it wasconfirmed that these λ's contained overlapping fragments of the genomeof Aspergillus niger var. awamori. Also homologous hybridization oftotal induced RNA with respectively λ-1, λ-14 and the 5.3 kb SAlIfragment of λ1-14 confirmed the presence of xyl A sequences on theseλ's. Hybridization was found with a xylan-induced mRNA of ca. 1 kb. Thesize thereof corresponds to that of the ARNA molecule hybridizing withXyl06.

1.2 Subcloning of the A. niger var. awamori xyl A Gene

The SalI fragments hybridizing with Xyl06 of respectively λ-1 (4.6 kb)and λ-14 (5.3 kb) were cloned in two orientations in the SalI site ofpUC19, which resulted in plasmid pAW1 (A and B) and plasmid pAW14respectively (A and B, see FIG. 3). The 1.2 kb PstI-BamHI fragmenthybridizing with Xyl06 and the adjacent 1.0 kb BAmHI-PstI fragment fromrespectively pAW14A and pAW1A were subcloned into M13mp18 and M13mp19cleaved with BamHI and PstI, resulting in the m18/m19 AW vectors ofTable C.

TABLE C Single-stranded subclones of λ-1 and λ-14 fragments fragmentresulting vectors pAW 1A BamHI-PstI* (1.2 kb) m18AW1A-1/m19AW1A-1 pAW14ABamHI-PstI* (1.2 kb) m18AW14A-1/m19AW14A-1 pAW 1A PstI-BamHI (1.0 kb)m18AW1A-2/m19AW1A-2 pAW14A PstI-BamHI (1.0 kb) m18AW14A-2/m19AW14A-2

1.3 Determination of the Transcription Direction of the xylA Gene

The transcription direction of the xylA gene was established by means ofspot blot hybridization of ss-DNA of respectively m18AW14A-1 andm19AW14A-1 with Xyl06(SEQ ID NO: 5). It was found that ss-DNA ofm19AW14A-1 (5′*PstI-BamHi 3′) hybridizes with this probe. Because thesequence of Xyl06(SEQ ID NO:5) is equal to that of the non-codingstrand, m19AW14A-1 contains the coding strand. On the basis thereof thetranscription direction shown in FIG. 2 was determined. This directionis confirmed by the results of a primer extension experiment.

1.4 Identification of the xylA Gene

The DNA sequence of a part of the promoter region was determined bysequence analysis of pAW14 with Xyl06 as a primer (5′ part of the gene).In this region a primer Xyl11(SEQ ID NO:6) with the sequence 5′- GCA TATGAT TAA GCT GC-3′ was selected, with which the DNA sequence ofcomplementary strand of m18AW14A-1 and m18AW1A-1 was determined. Theresults showed that these vectors contained a DNA sequence which wassubstantially equal to that of Xyl06(SEQ ID NO:5), while the amino acidsequence derived from the base pair sequence was identical to theN-terminal amino acid sequence of the mature xylanase protein. Thus thecloning of at least the 5′ end of the xylA gene was proved. The presenceof the whole xylA gene in the vectors pAW14 and pAW1 seemed plausible onthe basis of the position of the 5′ end of the gene on the SalIfragments (FIG. 2) and the size of the xylA mRNA (ca. 1 kb).

1.5 Sequence Analysis

The base sequence of the xylA gene was established in two directions inboth the m13AW14 and the m13AW1 subclones by means of the Sanger dideoxyprocedure (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467,1977). The sequence around the BamHI site located downstream of thePstI* site (FIG. 2) was established by sequence analysis ofdouble-stranded pAW14 and pAW1 DNA. Compressions are cleared up by usingdITP instead of dGTP. In the independent clones λ-1 and λ-14 anidentical xylA sequence is established. The complete (coding) sequenceof the pre(pro) xylanase gene is As shown in FIG. 1 (SEQ ID NO:7). Themature xylanase protein is preceded by a leader peptide of 27 aminoacids. Between the alanine residues at the positions 16 and 17 acleaving site is probably present for the signal peptidase. From thelength of the leader peptide it can be derived that a second processingsite is present in the protein. The cleaving of the band between Arg(27) and Ser (28) possibly takes place by a KEX2-like protease.

1.6 Localization of the Intron

In the xylA gene an intron of 49 or 76 bp (231-279 or 231-306, see FIG.1 (SEQ ID NO:7)) was predicted on the basis of the presence of sequencescorresponding to “donor” and “acceptor” sites of introns in Aspergilli.Definite proof of the absence of a 76 bp intron was obtained byisolation of a xylanase peptide with the sequence Tyr-Ser-Ala-Ser-Gly .. . This peptide can only be localized in the protein from position 302(see FIG. 1 (SEQ ID NO:7)).

1.7 Determination of the 3′ End of the xylA Gene

The position of the stop codon of the xylA gene (position 683 in FIG. 1(SEQ ID NO:7)) was derived from DNA sequence data. This stop codon wasconfirmed, since the amino acid sequence of a peptide is identical tothe C-terminal amino acid sequence derived from DNA sequence data(position 641-682 in FIG. 1 (SEQ ID NO: 7)).

1.8 Evaluation of DNA and Protein Data

On the basis of the above data the gene encoding a xylanase ofAspergillus niger var. awamori is cloned on a 5.3 kb SalI fragment. TheDNA sequence of the gene, the Position of the intron and the length ofthe mRNA were established. The established N-terminal amino acidsequence of the mature protein was fully confirmed by the DNA sequence.On the basis of the above data it can be concluded that the xylA geneencodes a protein of 211 amino acids and that the first 27 amino acidsare post-translationally removed. The amino acid sequence derived fromthe DNA sequence of the xylA gene demonstrates a high degree of homologywith the amino acid sequence of Bacillus pumilus (201 amino acids) andBacillus circulans (213 amino acids including signal) xylanases.

2 Expression Vectors

Three expression vectors were constructed containing the genomic xylAgene from the translation start including the xylA terminator. Thesevectors were derived from pAW14B (FIG. 3).

2.1 Vector pAW14S with Aspergillus niger var. awamori xylA Promoter

The vector pAW14S (FIG. 4) comprises a 5.3 kb chromosomal DNA fragmentof Aspergillus niger var. awamori on which the xylA gene is located withits own expression signals. Further a 5.3 kb fragment of Aspergillusnidulans on which the acetamidase (amdS) gene is located is present onthis plasmid. In pAW14S the amds and the xylA gene have the sametranscription direction.

2.2 Vector RAW14S-2 with

Aspergillus nidulans gpdA Promoter

The plasmid pAW14S-2 (FIG. 6) differs from pAW14S in that theAspergillus niger var. awamori fragment located upstream of the ATGcodon of the xylA gene is replaced by the constitutive expressionsignals (up to the ATC triplet) of the Aspergillus nidulansglyceraldehyde-3-phosphate dehydrogenase (gpdA) gene. In the plasmid theamdS and the xylA gene have the same orientation. The right connectionbetween the gpdA promoter and the ATG codon of the xylA gene wasobtained by means of a synthetic DNA fragment. During the constructionthe plasmid pAW14B-2 in which the amdS selection marker is absent wasalso obtained (FIG. 5).

2.3 Vector pAW14S-3 with Aspergillus niger var. niger glaA Promoter

The vector pAW14S-3 (FIG. 7) comprises the inducible expression signalsof the Aspergillus niger var. niger glucoamylase (a) gene up to the ATGcodon followed by Aspergillus niger var. awamori sequences, starting atthe ATG triplet of the xylA gene. In addition, this plasmid alsocomprises the Aspergillus nidulans amdS gene as a selection marker. TheamdS gene and the xylA gene have the same orientation.

By means of the amdS selection marker transformants with the three abovementioned plasmids can be obtained in which the vector, and consequentlyalso the (optionally hybrid) xylA gene, has been integrated into thegenome in a large number of copies, in order to increase the productionof the xylanase protein.

3 Transformation of Aspergillus

The transformation frequency of

Aspergillus niger var. awamori varied from 0.03 to 0.23 (AW)transformants per μg vector DNA. In total, this resulted in five AW14S(xylA promoter), forty AW14S-2 (gpdA promoter) and eight AW14S-3 (glaApromoter) transformants. In the continued investigation differenttransformants prove to have a deviating growth behaviour. One of them,AW14S #1, yielded properly sporulating (AW14S #1A) and poorlysporulating (AW14S #1B) colonies.

Transformation of Aspergillus niger var. niger N402 proceeded moreefficiently than that of Aspergillus niger var. awamori. With pAW14S,pAW14S-2 and pAW14S-3 0.3, 0.3 and 1 (AB) transformants were foundrespectively per mg DNA. Twenty pAB14S (xylA promoter), thirty pAB14S-2(gadA promoter) and sixteen pAB14S-3 (glaA promoter) transformants werestreaked.

Co-transformation of Aspergillus niger var. niger pyrG AB4.1 with pAW14Sand the Aspergillus niger var. niger pyrG gene in pAB4.1 resulted in 0.2transformants per μg pAW14S DNA when both markers were selected. Uponthe first selection for amds 2 transformants per μg DNA were found,while the frequency in the first selection for pyrG was Ca. 20 μg pAW14SDNA. It appeared that ca. 30% of the co-transformants (AB4.1-14S)possessed both markers. Six of them were analyzed further.

4 Analysis of Multicopy Transformants

4.1 Analysis of A. niger var. awamori “xylA” Transformants (AW14S) AfterCulturing in Medium with Xylan as an Inducer

After culturing AW14S transformants with xylan as an inducer thexylanase production level obtained in the medium after 10 days wassignificantly higher than with the wild-type Aspergillus niger var.awamori strain. Upon storage of the media at 4° C. the enzyme iscompletely stable. The production levels in xylan medium are listed inthe following Table D.

TABLE D Xylanase production levels (in 10³ U/ml) of AW14S transform-ants after various culturing periods in xylan medium at 25° C. No. 3days 10 days 1 28 58 2 21 56 3 20 31 4 25 58 5 8 22 wt 5 13

4.2 Analysis of Aspergillus niger var. niger N402 “xylA”(co)transformants (AB4.1-14S) After Culturing in Medium with Xylan as anInducer

The xylanase activity was determined in xylan medium of the host strainAspergillus niger var. niger pyrG AB4.1 and of seven AB14S-1 Pyr+co-transformants after 48 and 72 hours of culturing respectively (seeTable E). Aspergillus niger var. niger AB4.1 produces little xylanase(ca. 5000 U). For four out of seven co-transformants a high xylanaseactivity of ca. 30000 U was found. The other co-transformants producedsomewhat less xylanase.

TABLE E Xylanase production levels (in 10³ U/ml) of AB4.1-14S and AB14Stransformants after different culturing periods in xylan medium at 25°C. AB4.1-14S 48 hours 72 hours #1 36 36 #6 31 20 #12 29 23 #23 10 6 #4215 10 #44 21 30 #45 pyrG 22 18 AW.wt 3 7 N402 3 5 AB4.1 4 5

4.3 Characterisation of Overproduced Xylanase Enzyme

It can be derived from the highly increased xylanase activity in themedium of Aspergillus niger var. awamori and Aspergillus niger var.niger N402 multicopy “xylA” transformants that the cloned gene encodesxylanase from Aspergillus niger var. awamori and that the transformantsare capable of overproduction of active xylanase. The presence of thedesired product was shown by protein-chemical analysis of the medium ofAW14S #1A. One dominant protein was present in the medium. Theisoelectric point (pI) and the N-terminal amino acid sequence of thismain component were equal to those of purified xylanase from wild-typeAspergillus niger var. awamori. The pI value found corresponded to thevalue calculated for the mature protein of 184 amino acids for which thecomposition has been derived from the DNA sequence. In baking tests theproduced xylanase also proved to possess the desired properties.

4.4 Analysis of A. niger var. awamori (AW14S-2) and A. niger var. niger(AB14S-2) “gpdA” Transformants After Culturing in Rich Medium

Six AW14S-2 transformants were cultured in rich medium (Table F). Aftertwo to three days a xylanase activity varying from 15000 to 20000 U wasfound in the medium of three transformants, while the other threeproduced less than half said activity. The wild-type strain produces noxylanase in rich medium. In addition, ten AB14S-2 transformants weretested. Three of them produced ca. 11000 U xylanase after 40 hours,which level was maintained for at least up to 72 hours. The other fiveproduced less xylanase enzyme, while the activity in the medium of #21Bfell from 9000 to 0 U within 24 hours.

It was shown that the production maximum of the best producing AW14S-2and AB14S-2 transformants is in general reproducible. However, themaximum is not reached when the mycelium grows in large globules, whilea higher maximum was found (19000 instead of 11000 U) in one culture ofAB14S-2 #5 in duplicate. The production levels are listed in Table F.

The results show that it is possible to produce active xylanase by meansof a translation fusion of the gpdA promoter and the xylA gene. However,the production by means of both AW14S-2 and AB14S-2 transformantsregulated by the gpdA promoter in rich medium is lower than the xylanaseproduction of “xylA” transformants in medium with xylan.

TABLE F Xylanase production levels (in 10³ U/ml) of AW14S-2 and AB14S-2transformants after various culturing periods in rich medium at 25° C.AW14S-2 24 hours 48 hours 72 hours #1 <1 3 >3 #4 1 2 2 #10 11 15 13 #22Δ * 4 20 20 #36 3 7 7 #39 * 10 16 12 AB14S-2 24 hours 40 hours 48 hours66 hours 72 hours #2 4 4 9 #5 Δ 3 11 11 11 12 double 15 16 19 #7 Δ 1 5 55 4 #8 4 3 4 #11 <1 <1 1 1 0 #14 2 2 3 #16 Δ 3 10 10 11 10 #17 Δ 3 10 1110 10 #18 1 5 8 10 8 #21B 4 8 9 0 0 Δ maxima, found when repeating theculture; a culture of AB14S-2 #5 in duplicate gave a higher maximum. Inrich medium A. niger var. awamori and transformant AW14S #4 produce noxylanase.

4.5 Analysis of Aspergillus niger var. awamori (AW14S-3 and Aspergillusniger var. niger (AB14S-3) “glaA” transformants After Culturing inMedium with Starch as an Inducer

Some AW14S-3 transformants and the Aspergillus niger var. awamoriwild-type strain were cultured in starch medium (Table G). A xylanaseactivity of 67000 U/ml was found in the medium of one transformant after90 hours of culturing, while two other transformants produced up to36000 U/ml. The production maximum of six analyzed AB14S-3 transformantslies one day earlier than of AW14S-3 transformants. An activity of 51000U/ml was found in the medium of one transformant two others producedabout 43000 U/ml, after 63 hours of culturing. The results show that thetranslation fusion between the glaA promoter and the xylA gene iseffected in the right manner. Both AW14S-3 and AB14S-3 transformantsproduce substantially as much xylanase enzyme in starch medium,regulated by the glaA promoter, as “xylA” transformants in medium withxylan.

TABLE G Xylanase production levels (in 10³ U/ml) of “glaA” transfor-mants AW14S-3 and AB14S-3 after different culturing periods (hours) instarch medium at 25° C. 40 hour 63 hour 90 hour AW14S-3 #1 37 37 #2 4 1415 #4 8 31 36 #7 16 49 67 AB14S-3 #4 23 51 29 #5 19 37 21 #7 23 44 18 #817 28 32 #14 21 43 19 #16 6 21 10

4.6 Analysis of “xylA” Transformants After Culturing in Medium withWheat Bran

It is apparent from the results (Tables H and I) that the productionlevel observed for AW14S #4 when culturing in medium with wheat bran ishigher than that in xylan medium. A high production level was obtainedwith AB4.1-14S (#1 and #44) and AB14S (#5 and #14) transformants. Thexylanase activity obtained with said transformants was determined asbeing as high as 140000 U/ml. This means a considerable increase withrespect to the production in xylan medium (30000 U/ml). It furtherappears that the production level of these Aspergillus niger var. nigertransformants is also maintained upon prolonging the culturing period,as was found earlier with Aspergillus niger var. awamori “xylA”transformants in xylan medium.

4.7 Analysis of “gpdA” Transformants After Culturing in Medium withWheat Bran

AW14S-2 #22 and 139 produced up to 28000 U/ml xylanase. The AB14S-2 #5and #17 transformants produced relatively little xylanase (activity upto 15000 U/ml) with wheat bran, as was also found in rich medium. Theproduction levels are listed in Tables H and I.

4.8 Analysis of “glaA” Transformants After Culturing in Medium withWheat Bran

The tested AW14S-3 (#1 and #7) transformants produced up to 25000 and45000 U/ml xylanase respectively in medium with wheat bran, which forboth is ca. 60-65% of the values found in starch (Table I). With AB14S-3(#4 and #14) transformants, however, a higher production was determinedwith wheat bran than in starch. The production levels determined are 1.5times higher than in starch. A production of 72000 U/ml was obtainerwith AB14S-3. A value of 66000 U/ml was found with AB14S-3 #14 (TableI).

TABLE H Xylanase production levels (in 10³ U/ml) of some AW and AB“xylA” and “gpdA” transformants after various culturing periods inmedium containing bran at 25° C. 40 hours 63 hours 4 days 7 days 12 daysAw wt 1 2 16 17 AW14S #4 8 20 27 61 80 AB14S #14 6 74 114 126 122AB4.1-14S #1 17 87 123 135 145 AW14S-2 #22 17 22 22 34 33 AW14S-2 #39 1822 21 24 20 AB14S-2 #5 13 15 11 8 8 AB14S-2 #17 9 13 11 7 7

TABLE I Xylanase production levels (in 10³ U/ml) of AW and ABtransformants after various culturing periods in wheat bran medium at25° C. 2 days 3 days 4 days 7 days 9 days 14 days AW wt 2 8 10 11 N402wt 4 2 1 AW14S #1A 18 39 51 #4 34 67 76 76 AB14S #5 45 80 100 111 109118 #14 45 77 79 100 92 AB4.1-14S #1 95 90 73 #44 121 144 148 145AW14S-2 #22 22 29 29 #39 17 26 28 AB14S-2 #5 15 14 12 — #17 10 11 9 —AW14S-3 #1 18 26 25 #7 37 45 45 41 AB14S-3 #4 72 54 44 23 17 #14 64 6669 55 55 55

4.9 Evaluation of the Results

The results as summarized in Table A show that multicopy transformantsof Aspergillus niger var. awamori (AW14S) and Aspergillus niger var.niger (AB14S) are capable of overproduction of active xylanase afterinduction of their own xylA promoter with respectively xylan and wheatbran as an inducer. Expression of xylanase by multicopy transformants ofAspergillus niger var. awamori and Aspergillus niger var. niger N402with the xylA gene under control of the gpdA promoter (respectivelyAW14S-2 and AB14S-2) and the glaA promoter (respectively AW14S-3 andAB14S-3) indicates that xylanase can be produced in a wide range ofsubstrates. Variability in the productivities between the differenttransformants may be the result of differences in copy number and/or ofdifferences in the site of integration into the genome. Of course, thetesting conditions may also have a significant effect on the xylanaseproduction. For optimization of the production, however, preference willbe given to strains showing a relatively high productivity.

5 Materials and Methods

5.1 Strains and Plasmids

In the experiments the following strains and plasmids were used:

Aspergillus niger var. awamori strain CBS 115.52, ATCC11358;

Aspergillus niger var. niger strain N402, a cspA1 (short conidiophores)mutant of Aspergillus niger var. niger ATCC9029, CBS 120.49;

Aspergillus niger var. niger AB4.1, a pyrG mutant of Aspergillus nigervar. niger N402, described by Van Hartingsveldt et al., Mol.Gen.Genet.206, 71-75, 1987;

Escherichia coli strain JM109 (for plasmid isolation, see Yanisch-Perronet al., Gene 33, 103-119, 1985);

Escherichia coli strain NM539 (for construction and amplification of thelambda-gene library);

plasmid pGW325, containing the amds gene of Aspergillus nidulans see K.Wernars, “DNA-mediated transformation of the filamentous fungusAspergillus nidulans”, Thesis, Agricultural University of Wageningen,1986;

plasmid pAB4.1, containing the p=gene of Aspergillus niger var. nigerN402, see Van Hartingsveldt et al., Mol. Gen.Genet. 206,71-75, 1987;

plasmid pAN52-1, described by Punt at al., Gene 56, 117-124, 1987; andplasmid pAN52-6, described by P. J. Punt, J. Biotechn., in press;

vector λ-EMBL3 (for construction of an Aspergillus niger var. awamorigene library), obtainable from Promega Biotec.

An Escherichia coli JM109 strain containing the plasmid pAW14B wasdeposited with the Centraalbureau voor Schimmel-cultures (CBS) of Baarn,The Netherlands, under number CBS 237.90, on May 31, 1990.

5.2 Aspergillus Transformation

Aspergillus niger var. awamori protoplasts were made from mycelium bymeans of Novozym 234 (NOVO). The yield of protoplasts was 1-5×107/gmycelium and the viability was 3-8%. Per transformation 3-8×105 viableprotoplasts were incubated with 5, 10 or 20 mg, plasmid DNA that hadtwice undergone CsCl purification. Transformed protoplasts were platedon osmotically stabilized selection plates (acetamide as a nitrogensource) and incubated at 25° C. After 6-10 days colonies were visible.Transformation of Aspergillus niger var. niger N402 and A. niger var.niger AB4.1 respectively was in principle carried out as describedabove. In the case of Aspergillus niger var. niger pyrG AB4.1, however,uridine was added to the medium. In the co-transformation of A. nigervar. niger AB4.1, pAW14S and pAB4.1 DNA were mixed in a weight ratio of4:1; transformants were selected on acetamide plates with uridine (amdSselection), without uridine (amdS and pyrG selection) and on minimalmedium plates with nitrate (pyrG selection) respectively. After 4-5 dayscolonies became visible. (Co-)transformants were streaked twice ontoacetamide plates. In order to obtain large amounts of spores, sporesfrom the second streak were streaked through onto plates with richmedium and incubated for 5-6 days at 25-28° C. The resulting spores werestored as a suspension (108-109 spores/ml) or adsorbed to silica gel sothat the spores can be stored for a long time.

5.3 Construction of A. niger var. awamori Gene Library

Chromosomal DNA was isolated from myceliun of Aspergillus niger var.awamori. The high molecular DNA was partially cleaved with Sau3AI,followed by isolation of fragments of 13-17 kb After electrophoresis ona 0.4% agarose gel. Of these fragments 0.4 mg were ligated with 1.2 mgλ-DML3 DNA which was cleaved with BamHI and EcoRI. The ligation mixturewas provided with phage coats by means of an in vitro packing system(Amersham). By transduction to E. Coli NM539 a gene library of ca. 154000 plaques was obtained. These presented ca. 75×the genome of A. nigervar. awamori. 65000 Plaques were transferred to nitrocellulose filters(in plicate).

5.4 Hybridisation Experiments

Southern blot analysis: hybridisation of digests of chromosomal A. nigervar. awamori DNA with radioactively labelled oligonucleotide mixturesXyl04 and Xyl06 (SEQ ID NO:s 3 and 5) (47 mers) was carried out in 6×SSCat respectively 68° C., 62° C. and 56° C.; for Xyl01 and Xyl05 (SEQ IDNO:s 2 and 4, respectively) (23 mers) a hybridization temperature of 41°C. was used. The selected hybridization temperature was at least 5° C.below the calculated melting temperature. Blots were washed at thehybridization temperature with 5×and 3×SSC respectively. Hybridizationwas carried out at 68° C. in 6×SSC, while the last washing steps werecarried out at the same temperature with 2× and 0.4×SSC respectively.

Northern blot analysis: total, non-induced RNA of A. niger var. awamoriwas isolated from mycelium of rich medium cultures (after 3 days ofculturing at 25° C.). Induced RNA originated from cultures in which 1%xylan or 4% wheat bran was used as an inducer. Mycelium was collectedfrom the last-mentioned cultures after different culturing periods.After 3 and 6 days respectively mycelium was isolated from medium withwheat bran. Mycelium of xylan medium was collected after 6 and 11 daysof culturing respectively. Hybridization conditions were equal to thosein the Southern blot analysis.

5.5 Culturing Conditions

Media: xylan medium contains 1% xylan, 0.67% yeast extract with aminoacids (Difco) and 0.1% cas. amino acids. Medium with wheat bran consistsof 4 g wheat bran in 50 ml mains water, to which 50 ml of a saltsolution (pH 5.0) is added up to a final concentration of 0.5%(NH4)2SO4, 0.15% KH2PO4, 0.025% MgSO4 AND 0.025% KCl. Rich medium forexpression tests is minimal medium (0.05% MgSO4, 0.6% NaNO3, 0.05% KCl,0.15% KH2PO4 and trace elements), with 1% glucose, 0.2% trypticase(BBL), 0.5% yeast extract, 0.1% cas. amino acids and vitamin. Starchmedium contains 5% starch and 0.1% glucose in minimal medium. Media weresterilized for 30 min. at 120° C. Medium (100 ml in a 500 ml flask) wasinoculated with 2×105 spores/ml, followed by culturing in an airincubator (300 rpm) at 25° C. for different periods. Cultures with wheatbran as an inducer (Table I) were inoculated with 4×105 spores/ml.

5.6 Determination of Xylanase Activity in Medium of Aspergillus Cultures

The xylanase activity was established by determining the formation ofreducing sugars. Procedure: a (diluted) medium sample was added to 125μl 2% xylan (Sigma) in 0.5 M Na acetate pH 5.0 at 40° C., followed byincubation of the reaction mixture for 30 min. at 40° C. The reactionwas immediately stopped with 0.5 ml 2-hydroxy-3,5-dinitro-benzoic acid(DNS) reagent, followed by supplementing the volume with water up to 1ml. The reaction mixture was heated for 5 min. at 100° C. and cooled toroom temperature. The OD was determined at 534 nm against a blank. Thexylanase activity determination of one sample was carried out at leasttwice. 0.5 M Na acetate pH 5.0 was used for diluting the media

5.7 Selection of Transformants

In the analysis of the many transformants the host strains and ca. 6transformants from one series were cultured in rich or selective medium,followed by determination of the xylanase production level. Twotransformants from each series, with the highest xylanase production,were analyzed again in the same medium. In addition, the productionlevel of these transformants was determined in medium with wheat bran.

5.8 Construction of Expression Vectors

pAW14S (with the Aspergillus niger var. niger xylA promoter): theexpression vector pAW14S (FIG. 4) was constructed by insertion of a 5.0kb EcoRI fragment of plasmid pGW325, on which the Aspergillus nidulansamdS gene is located, into the EcoRI site of the polylinker of pAW14B(FIG. 3). In pAW14S the amdS and xylA gene have the same transcriptiondirection.

pAW14S-2 (with the A. nidulans gpdA promoter): the linear 1.8 kbStuI-NcoI fragment of pAN52-1, on which the A. nidulans gpdA promoter(up to the ATG triplet) is located, was ligated with the 7.2 kbNcoI*-SmaI fragment of pAW14B, obtained by partial digestion with NcoIand complete digestion with SmaI. Transformation of E. coli JM109resulted in isolation of plasmid pAW14B-1 (9.0 kb1). The 7.2 kbNruI*-NcoI* fragment of pAW14B-1, obtained by partial digestion withNcoI and complete digestion with NruI, was ligated with a syntheticfragment (79 bp, nucleotides Nos. 1-78 of the coding strand andnucleotides Nos. 4-78 of the template strand), consisting of xylAsequences from the ATG triplet, resulting in pAW14B-2 (FIG. 5). The 5.0kb EcoRI fragment of pGW325 (Aspergillus nidulans amdS gene) wasintroduced into the unique EcoRI site of pAW14B-2, resulting in pAW14S-2(FIG. 6). The amdS and the xylA gene have the same orientation in thisplasmid. The connection of the gpdA promoter to the ATG codon of thexylA gene as well as the sequence of the synthetic fragment was verifiedby means of DNA sequence analysis.

pAW14S-3 (with the A. niger var. niger N402 glaA promoter): pAN52-6 waspartially cleaved with XmnI (3 sites). The linear 7.5 kb fragment, onwhich the A. niger var. niger N402 glaA promoter is located, wasisolated. After cleaving this fragment with BssHII a 7.35 kb BssHII-XmnIfragment was ligated with a synthetic DNA fragment (ca. 150 bp)containing the 3′ end of the alaA promoter up to the ATG triplet,followed by the xylA gene from the ATG triplet to the NruI site locatedin the gene with a BssHII terminus behind that. The plasmid pAN52-6.URLthat was thus obtained was cleaved with NcoI and with NruI, afterfilling in the NcoI site. The DNA sequence of the synthetic fragment inpAN52-6.URL was checked. The glaA promoter was placed before the xylAgene by ligation of the 2.5 kb “filled NcoI”-NruI fragment frompAN52-6.URL with the ca. 10 kb NruI fragment of pAW14S. Insertion ofthis fragment in the right orientation resulted in pAW14S-3 (FIG. 7).

EXAMPLE II

Baking Tests

The bread improving activity of the xylanase, obtained after isolationfrom fermentation broth, was tested by measuring the volume increase ofBelgian bread rolls baked after addition of increasing amounts of enzymeand dough. The xylanase was isolated as follows.

Aspergillus niger var. awamori transformant AW14S.1A was cultured for 7days on medium with 4% wheat bran in a fermentor having an operatingvolume of 8 liter. The xylanase production was ca. 85000 U/ml. Thefungal cells were removed by a filtration over a cloth. Ammoniumsulphate was then added to 6 liters of filtrate, with stirring, up to50% by weight. The precipitate was centrifuged in a Sorvall GSA rotor at10000 g for 20 minutes. The pellet was suspended in 500 ml aqua dest.and then centrifuged again at 10000 g. The supernatant was thenconcentrated by ultrafiltration by means of an Amicon PM10ultrafiltration membrane up to a volume of 60 ml. In order to remove theammonium sulphate the ultrafiltration was repeated twice after dilutionwith aqua dest. to 300 and 600 ml respectively. The finally obtainedmaterial that was present in a volume of 50 ml was then freeze dried.The yield was 4.8 g with a specific activity of 60000 U/mg (56%overall). For use in baking tests the xylanase was mixed with starch toa concentration of 240 U/mg.

600 ml Water, 20 g salt, 20 g sugar (sucrose), 50 g yeast (Koningsgistfrom Gist Brocades) and 0, 50, 100 or 200 mg/kg xylanase (240 U/mg) wereadded to 1000 g wheat flour Banket Extra (from Wessanen). The doughspecies were kneaded in an Eberhardt kneader for ten minutes at a doughtemperature of 24° C. After 20 minutes of fermentation at 28° C. thedough was beaten, divided into small dough portions of ca. 50 g and onceagain fermented in a raising cabinet for 60 minutes at 35° C. to 38° C.The dough portions were then baked at 230° C. for 20 minutes. Thespecific volumes (in ml/g) were determined by dividing the volume (inml), determined by means of the seed displacement method, by the weight(in g).

For an average of 10 bread rolls the following results were found:

enzyme level 0 50 ppm 100 ppm 200 ppm specific volume 6.8 7.9 8.7 8.9

The same trends can be established if, moreover, other bread improvingingredients such as vitamin C, fat, emulsifiers and α-amylase are added.Other properties such as dough processing and crumb structure are alsopositively affected by adding the xylanase enzyme.

EXAMPLE III

Production of Aspergillus niger var. awamori Xylanase by Saccharomycescerevisiae

As an example of the heterologous production of Aspergillus niger var.awamori xylanase by microorganisms, expression vectors were constructedfor the expression of the xylanase in Saccharomyces cerevisiae regulatedby the inducible GAL7 promoter (Nogi and Fukasawa, 1983). The GAL7promoter effects production of enzyme under inducing conditions: growthon medium with galactose as the only carbon source (Hopper and Rowe,1978). The use of this promoter for induced production of heterologousproteins has already been described (Tajima et al., 1985). The fungalgene encoding xylanase was first rendered suitable for expression inSaccharomyces cerevisiae by removing the intron (non-coding sequence) bymeans of a synthetic DNA fragment. The same technique has been used toprovide a correct connection of the xylanase gene to the Saccharomycescerevisiae GAL7 promoter. Optionally, a Saccharomyces cerevisiae signalsequence, the invertase signal sequence, was also introduced to realizethe secretion of the fungal enzyme xylanase by the yeast Saccharomycescerevisiae. Autonomously replicating vectors as well as (multicopy)integrating vectors have been used in the production of the Aspergillusniger var. awamori xylanase by the yeast Saccharomyces cerevisiae. Allcloning procedures were carried out in E. coli strain JM109(Yanisch-Perron et al., 1985) and all methods and techniques accordingto Maniatis et al. (1982).

Construction of Vector pUR2901

The first intermediate construction was directed at the correct removalof the intron from the xylanase gene, i.e. without changing ordisturbing the coding sequence. The synthetic DNA oligonucleotides shownin FIG. 8 (BAK 02, 03, 04, 05, 06, 07, 08, 09, 10, 23 and 24 (SEQ NO IDNO:s 34-37,43,42,41,40,39,33 and 38, respectively)) were annealed andligated together resulting in the fragment BAK1(SEQ ID NO:9). Thefragment BAK1 measures 205 bp and comprises the SacI—KpnI xylanasefragment (bp 185-bp 427) from which the intron has been removed. Thesynthetic DNA oligonucleotides have been designed in such a manner thatupon removal of the intron a correct connection to the fragments hasbeen made so that the open reading frame (encoding xylanase) is notdisturbed. In order to simplify the continued construction the SacI sitewas changed to an XhoI site. On the 5′ side the fragment was providedwith an EcoRI site. The ligation mixture was digested with therestriction enzymes KpnI and EcoRI and the correct 205 bp fragment wasisolated by means of agarose gel electrophoresis for the separation ofthe fragment and gel elution for the isolation of the fragment from theagarose gel. The KpnI-EcoRI BAX1 fragment was cloned into the KpnI andthe EcoRI site of vector pTZ19R (obtained from Pharmacia) resulting inpBAK1 (see FIG. 9). The inserted fragment in the constructed plasmidpBAK1 was checked by means of sequence analysis.

The continued constructions were directed at the realization of acorrect connection of the Aspergillus niger var. awamori xylanase geneto the Saccharomyces cerevisiae GAL7 promoter. For this purpose thesynthetic DNA oligonucleotides shown in FIG. 10 (BAK13, 14, 15, 18, 19,20, 21, 25, 26, 27 and 28 (SEQ ID NO:s 17-19, 25,24, 23,22,21,20,26 and21, 39 respectively) were annealed and ligated resulting in fragmentBAK2 (SEQ ID NO:10). Fragment BAK2 measures 202 bp and comprises thesynetic transition from the SacI site of the GAL7 promoter via theinvertase signal sequence to the mature xylanase gene up to the SacI (bp185) site. In order to simplify the continued construction the SacI sitewas changed to an XhoI site, in a manner identical to the one used inthe construction of pBAK1. An additional EcoRI site was provided on the5′ side of the fragment. The ligation mixture was digested with EcoRIand XhoI and the correct 202 bp BAK2 fragment was isolated. PlasmidpRAK1 was digested with EcoRI and XhoI and the BAK2 (SEQ ID NO:10)fragment with the same termini was cloned in the vector fragments,resulting in plasmid pBAK21 (see FIG. 11). The inserted BAK2 (SEQ IDNO:10) fragment was checked by means of sequence analysis. In plasmidpBAK21 the connection of fragments BAK1 and BAK2 (SEQ ID NO:s 9 and 10,respectively) to the XhoI site was effected in such a manner that theopen reading frame encoding xylanase was correctly restored. PlasmidpBAK21 therefore contains the Saccharomyces cerevisiae GAL7 promotertransition from the SacI site, the Saccharomyces cerevisiae invertasesignal sequence (including an ATG start codon) and the Aspergillus nigervar. awamori xylanase (encoding mature xylanase) from which the fungalintron (non-coding sequence) has been correctly removed up to the KpnIsite (the 5′ part of the xylanase gene).

Plasmid pAW14B was digested with KpnI and BamHI and the 327 bpKpnI-BamHI fragment, containing the 3′ part of the xylanase gene, wasisolated. Plasmid pBAK21 was also digested with KpnI and BamHI and thevector fragment was isolated. The isolated 327 bp fragment and thevector fragment were ligated together resulting in plasmid pUR2901 (seeFIG. 12). Plasmid pUR2901 was checked by means of restriction enzymeanalysis. Plasmid pUR2901 contains the S. cerevisiae GAL7 promoterfusion site at the SacI site, the S. cerevisiae invertase signalsequence (including an ATG start codon), and the complete Aspergillusniger var. awamori xylanase gene (encoding mature xylanase) from whichthe fungal intron (non-coding sequence) has correctly been removed.

Construction of the S. cerevisiae Expression Vector pUR2904

The construction of expression vector pUR2904 started from plasmidpUR2740. Plasmid pUR2740 is a derivative of pUR2730 (Overbeeke 1987)used for the production of α-gal-actosidase in S. cerevisiae. PlasmidpUR2740 is not essentially different from pUR2730, some superfluoussequences in the non-functional part of the vector have been removed.Plasmid pUR2740 is an E coli/S. cerevisiae shuttle vector. Use was madeof the 2 μm origin of replication, and the S. cerevisiae LEU2d geneserved as a selection gene for the replication in S. cerevisiae. PlasmidpUR2740 was digested with SacI and HindIII, and the vector fragment wasisolated. As a result of this digestion, the α-galactosidase gene wasremoved. Plasmid pUR2901 was also digested with SacI and HindIII, andthe 730 bp fragment comprising the S. cerevisiae GAL7 promoter fusionsite at the ZacI site, the S. cerevisiae invertase signal sequence(including an ATG start codon), and the complete A. niger var. awamorixylanase gene (encoding mature xylanase) was isolated. The pUR2740vector fragment and the 730 bp fragment of pUR2901 were ligatedtogether, resulting in pUR2904 (see FIG. 13). Plasmid pUR2904 waschecked by means of astriction enzyme analysis. Plasmid pUR2904 is theexpression vector for the production of the Aspergillus niger var.awamori xylanase by the yeast Saccharomyces cerevisiae Plasmid pUR2904is an E. coli/S. cerevisiae shuttle vector. It contains the DNA sequenceencoding xylanase with the invertase signal sequence fused to it; theinvertase signal sequence will provide the secretion of the xylanase.The DNA sequence in pUR2904 encodes exactly the same xylanase as thewild-type A. niger var. awamori strain. During secretion the resultingfusion protein will, in principle, undergo processing by theSaccharomyces cerevisiae signal peptidase resulting in secreted maturexylanase enzyme. The expression of the xylanase is regulated by theSaccharomyces cerevisiae galactose inducible GAL7 promoter.

Analysis of the Production of A. niger var. awamori Xylanase by S.cerevisiae

Yeast cells of the Saccharomyces strain SU10 (α, leu2, ura3, his3, cir+;deposited with the Centraalbureau voor Schimmelcultures, P.O. Box 273,3740 AG Baarn, The Netherlands, under number CBS 323.87) weretransformed with plasmid pUR2904 via the spheroplast method (Beggs,1978). The resulting leu+ transformed yeast cells were analyzed for thepresence of xylanase. The yeast cells were twice grown overnight on MMmedium (0.67% Yeast Nitrogen Base w/o amino acids, 2% glucose)supplemented with uracil and histidine. Subsequently, the yeast cellswere transferred to a ten times larger volume of YPG medium (1% YeastExtract, 2% Bacto peptone, 5% galactose) and grown until the yeast cellshad reached the stationary phase. The yeast cells were cultured underagitation at 30° C. The yeast cells were separated from the medium bycentrifugation. The medium was analyzed for the presence of the xylanasewith the enzyme assay as described in Example I. The expression level ofxylanase was about 10000 units in 1 ml medium. By means of isoelectricfocussing (see Example I) it was demonstrated that the xylanase producedby Saccharomyces cerevisiae is identical to the xylanase produced bywild-type Aspergillus niger var. awamori. The functionality of thexylanase, produced and secreted by Saccharomyces cerevisiae was shown inbaking tests carried out as described in Example II. The resultsdescribed above show that the yeast Saccharomyces cerevisiae is capableof efficiently producing and secreting Aspergillus niger var. awamorixylanase.

Construction of the S. cerevisiae Expression Vector PUR2921 (multi-copyIntegration)

The expression of the xylanase gene of Aspergillus niger var. awamori inSaccharomyces cerevisiae was also studied by an integrative vectorsystem. For this purpose the high-copy integration system was used(Lopes, 1989).

The construction of expression vector pUR2921 started from plasmidpUR2778. Plasmid pUR277 is a multi-integrative plasmid integrating inthe ribosomal DNA locus of S. cerevisiae. It was used for stablemulti-copy integration of the α-galactosidase expression cassette in S.cerevisiae. It also contains vector sequences for replication andselection in E. coli and the S. cerevisiae LLU2d gene as a selectiongene for yeast. Plasmid pUR2778 is a derivative of pMIRY2 (Lopes, 1989),from which the SmaI-BgIII fragment containing the Spirodella oligorhizaDNA has been removed, and the BamHI-HindIII fragment containing a partof the rDNA sequences has been replaced by the BglII-HindIII fragment ofpUR2730 (Overbeeke, 1987) containing the α-galactosidase expressioncassette. Plasmid pUR2778 was digested with SacI and HindIII, and thevector fragment was isolated from agarose gel. As a result of thisdigestion the α-galactosidase coding sequence including the invertasesignal sequence was removed. This vector fragment was ligated with the730 bp SacI-HindIII fragment from pUR2901, which was also used for theconstruction of pUR2904, resulting in plasmid pUR2921 (see FIG. 14). The730 bp SacI-HindIII fragment of pUR2901 comprises the S. cerevisiae GAL7promoter fusion site at the SacI site, the S. cerevisiae invertasesignal sequence (including ATG start codon), and the complete A. nigervar. awamori xylanase gene (encoding mature xylanase) from which thefungal intron (non-coding sequence) has correctly been removed. PlasmidpUR2921 was checked by means of restriction enzyme analysis. PlasmidpUR2921 is an expression vector for the production of the Aspergillusniger var. awamori xylanase by the yeast Saccharomyces cerevisiae.Plasmid pUR2921 contains sequences of the ribosomal DNA locus of the S.cerevisiae chromosomal DNA. As it does not contain any yeast replicationorigins the vector will integrate at the ribosomal DNA locus upontransformation to S. cerevisiae. When the pUR2921 plasmid is transformedto a S. cerevisiae leu2 strain, under selective conditions multiplecopies of the vector will integrate, due to the low expression of theLEU2 marker gene of the pUR2921 plasmid. As a result of this process,the xylanase expression cassette will be present in multiple copies inthe yeast chromosome. As the xylanase expression cassette is exactly thesame as in the pUR2904 plasmid, this S. cerevisiae strain will secretethe mature xylanase enzyme in the same way as the S. cerevisiae strainwith the pUR2904 plasmid.

Analysis of the Production of A. niger var. awamori Xylanase by S.cerevisiae

Yeast cells of the Saccharomyces strain SU50 (YT6-2-1, a, leu2, his4,can1, cir°; Erhart and Hollenberg, 1981) were transformed by thespheroplast method with plasmid pUR2921, linearized with HpaI. Theresulting leu+ transformed yeast cells were analyzed for xylanaseproduction as described for the SU10 yeast cells transformed with thepUR2904 plasmid. For these yeast cells the MM medium was onlysupplemented with histidine. The expression level was about 60,000 unitssecreted in 1 ml medium.

REFERENCES

Beggs, J. D. (1978), Nature 275: 104-109.

Erhart, Hollenberg (1981), Curr. Genet. 3:83-89.

Hopper, J. E. and Rowe, L. B. (1978), J. Biol. Chem. 253:7566-7569.

Lopes, T. S., Klootwijk, J., Veenstra, A. E., van der Aar, P. C., vanHeerikhuizen, H., Raué, H. A. and Planta, R. J. (1989), Gene 79:199-206.

Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982), Molecular Cloning.A laboratory manual, Cold Spring Harbor Laboratory.

Nogi, Y. and Fukasawa, T. (1983), Nucleic Acids Res. 11:8555-8568.

Overbeeke, N., Fellinger, A. J. and Hughes, S. G. (1987), PCTInternational. WO 87/07641.

Tajima, M., Nogi, Y. and Fukasawa, T. (1985), Yeast 1:67-77.

Yanisch-Perron, C., Viera, J. and Messing, J. (1985) Gene 33:103-119.

EXAMPLE IV

Production of Aspergillus niger var. awamori Xylanase by Bacillussubtilis

As an example of the heterologous production of Aspergillus niger var.awamori xylanase by a prokaryotic microorganism, expression vectors wereconstructed for the production of xylanase by Bacillus subtilis. Variousvector systems, promoters and signal sequences are known for theproduction of heterologous proteins. For this example the SPO2 promoterand α-amylase signal sequence were used for the expression of thexylanase enzyme. This approach has been successful for the expression ofthe plant α-galactosidase in B. subtilis (Overbeeke et al, 1990).

For the construction of a vector for the expression of Aspergillus nigervar. awamori xylanase by Bacillus subtilis, plasmids constructed for theexpression of xylanase in Saccharomyces cerevisiae (see example III), inwhich the intron (non-coding sequence) of the xylanase gene wascorrectly removed, were used as a starting point. Removal of the intronis essential, because a prokaryotic microorganism such as Bacillussubtilis is not capable, unlike the eukaryote Aspergillus niger var.awamori, of removing introns by a process called splicing.

Construction of Vector pUR2950

The synthetic DNA oligonucleotides shown in FIG. 15 (BAX 15, 18, 26, 27,41 and 42(SEQ ID NO:s 19, 25,20,26,28 and 27, respectively)) wereannealed and ligated together resulting in fragment BAX4(SEQ ID NO:11).Fragment BAK4 (SEQ ID NO:11) measures 107 bp and comprises the DNAsequence encoding mature xylanase up to the SacI site (bp 185 in FIG.1). In order to simplify the continued construction, the SacI site waschanged to a XhoI site without changing the derived amino acid sequence.Moreover, in order to obtain a correct connection of the mature xylanaseto the α-amylase signal sequence in the continued construction, thefirst codon of the mature xylanase encoding alanine was changed. Thecodon GCT was changed to GCC, also encoding alanine. Thus a SacII sitewas created on the 5′ side of the BAK4 (SEQ ID NO:11) fragment.Continuous with this SacII site the BAK4 (SEQ ID NO:11) fragment wasprovided with an EcoRI site. The ligation mixture was digested withEcoRI and XhoI and the 107 bp EcoRI-XhoI fragment was isolated fromagarose gel. Plasmid pUR2901 (see example III) was digested with EcoRIand XhoI and the BAK4 (SEQ ID NO:11) fragment was cloned in the vectorfragment with the same restriction enzyme termini, resulting in pUR2950(see FIG. 16). The inserted fragment BAK4 (SEQ ID NO:11) pUR2950 waschecked by means of sequence analysis. In plasmid pUR2950 the connectionof fragment BAK4 (SEQ ID NO:s 11 and 9, respectively) and BAK1 by meansof the XhoI site was carried out in such a manner that the open readingframe encoding the 5′ part of xylanase was correctly restored. Moreover,as described in Example III, the intron of the xylanase gene wascorrectly removed. Accordingly, plasmid pUR2950 contains the DNAsequence from the first alanine codon of mature xylanase, in which aSacII site has been made at that location, and the Aspergillus nigervar. awamori xylanase gene encoding mature xylanase from which thefungal intron (non-coding sequence) has been correctly removed.

Construction of B. subtilis Expression Vector pUR2951

Plasmid pUR2601 (Overbeeke, 1990) was used as a base for theconstruction in which the mature Aspergillus niger var. awamori xylanasegene present in pUR2950 has been fused to the α-amylase signal sequencefor secretion of the enzyme to be produced, while this fusion gene isregulated by the SPO2 promoter. Plasmid pUR2601 was digested with SacIIand HindIII and the vector fragment with the SPO2 promoter and theα-amylase signal sequence was isolated. The SacII-HindIII fragment ofpUR2950 with the mature xylanase gene was isolated and ligated to thepUR2601 vector fragment, resulting in plasmid pUR2951 (FIG. 17). Inplasmid pUR2951 the α-amylase signal sequence has been fused in exactlythe correct manner to the mature xylanase gene. The ligation mixture wastransformed to the Bacillus subtilis strain DB104 (Kawamuri and Doi,1984) using the protoplast/PEG method (Chang and Cohen, 1979) withkanamycin for selection. The plasmid pUR2951 was checked withrestriction enzyme analysis.

Analysis of the Production of A. niger var. awamori Xylanase by B.subtilis

Because the DB104 strain has some residual protease activity,fermentation of DB104 with pUR2951 under controlled conditions isnecessary to avoid proteolysis of the secreted enzyme. The approachdescribed by Overbeeke et al. (1990) for the production of plantα-galactosidase by B. subtilis, can be used as starting point for theproduction of Aspergillus conditions is necessary to avoid proteolysisof the secreted enzyme. The approach described by Overbeeke et al.(1990) for the production of plant α-galactosidase by B. subtilis, canbe used as starting point for the production of Aspergillus niger var.awamori xylanase by Bacillus subtilis. In this fermentation specialattention is paid to the glucose and ammonium levels duringfermentation.

REFERENCES

Chang, S., and Cohen, S. N. (1979), Mol. Gen. Genet. 182:77-81.

Kawamura, F., and Doi, R. H (1984), J. Bacteriol. 160:442-444.

Overbeeke, N., Termorshuizen, G. H. M., Giuseppin, M. L. F., Underwood,D. R., and Verrips, C. T. (1990), Appl. Environ. Microbiol.56:1429-1434.

EXAMPLE V

Production of Aspergillus niger var. awamori Xylanase by Saccharomycescerevisiae During Fermentation of Lean Bread Dough

As an example of the direct use of cells which produce the Aspergillusniger var. awamori xylanase in foodstuffs, a Saccharomyces cerevisiaestrain which produces the xylanase during fermentation of lean breaddough has been constructed. For this purpose the S. cerevisiae strainmust secrete the xylanase under conditions present during thefermentation of wheat dough. In lean wheat dough no sugar is added andtherefore the main carbon sources for the fermenting bakers yeast areglucose and maltose. The xylanase gene should therefore be regulated bya promoter which is not affected by glucose repression. Promoters of thegenes of the glycolytic pathway (GAPDH, PGK, ADH1, PYK, etc.) of yeastare extremely useful for this purpose. Just by way of example thepromoter of the phosphoglycerate kinase (PGK ) gene of Saccharomycescerevisiae was used. This promoter is used for the expression by S.cerevisiae of numerous heterologous proteins, for example the productionof human interferon-alpha (Tuite et al., 1982).

The preferred way to achieve the expression of an enzyme during breadmaking is to use an integrative vector (single-copy or multi-copyintegration), although autonomously replicating vectors can also beused.

The GAL7 promoter from the pUR2921 plasmid, used for the expression ofthe Aspergillus niger var. awamori xylanase in Saccharomyces cerevisiae(see example III), was replaced by the PGK promoter. S. cerevisiaestrains transformed with this new vector, and which secretes thexylanase enzyme in culture media containing glucose, could be used inbread making. Addition of this yeast to dough before mixing results insecretion of the xylanase enzyme during bread making, and thus exhibitsthe positive effect of this bread improving enzyme, resulting in anincreased specific volume of the bread.

Construction of Plasmid pUR2918

For the fusion of the Saccharomyces cerevisiae PGK promoter sequences tothe Aspergillus niger var. awamori xylanase gene several approaches werepossible. Among others the creation of a suitable restrictionendonuclease site at the end of the promoter by means of site directedmutagenesis that could yield a DNA molecule which, for example, could befused to the SacI site between the GAL7 promoter and invertase signalsequence of pUR2904, the plasmid used for the expression of the maturexylanase by Saccharomyces cerevisiae. Another way of creating suitablerestriction sites at the end of a DNA molecule is by way of an in vitroamplification technique of DNA known as Polymerase Chain Reaction. ThisPCR technique was used to generate a DNA molecule containing allimportant sequences of the Saccharomyces cerevisiae phosphoglyceratekinase promoter up to the ATG startcodon, with an EcoRI and a BglII siteat its 5′ end and a BspMI recognition sequence and a HindIII site (seeFIG. 18 (SEQ ID NO: 12)) 3′ to the ATG start3codon. The primers used forthe amplification were PGP01 (SEQ ID NO: 13): 5′-GA ATT CAG ATC TTG AATTGA TGT TAC CCT CAT AAA GCA CGT G-3′ and PGP02 (SEQ ID NO: 14): 5′-CCCAAG CTT ACC TGC TGC GCA TTG TTT TAT ATT TGT TGT AAA AAG TAG ATA ATT ACTTCC-3′. Template DNA was pUR2802, a yeast expression vector with thecomplete Saccharomyces cerevisiae PGK promoter. The reaction mixture(total volume 100 μl) was composed as follows: Aprox. 1 ng pUF2801cleaved with SalI, 100 pmoles of PGP01 (SEQ ID NO: 13) and 100 pmoles ofPGP02, 1 U Amplitaq polymerase (Perkin Elmer), 0.2 mmol/l of each dNTP:dATP, dCTP, dGTP and dTTP, 1.5 mmol/l MgCl₂, 50 mmol/l KCl, 10 mmol/lTris HCl pH 8.3 (at 25° C.), 0.001% (w/v) gelatine. After 2 minutes ofincubation at 95° C., 25 cycles of the following temperature steps werecarried out: 1 min at 95° C., 1:45 min at 52° C., 2 min at 72° C. Afterthese cycles the reaction mixture was maintained at 72° C. for 5 minbefore cooling to 4° C. All temperature cycles were performed in aPerkin Elmer DNA Thermal Cycler. 60 μl was precipitated from thereaction mixture with ethanol and subsequently the band of approximately600 bp was isolated from agarose gel. The isolated DNA was then cleavedwith EcoRI and HindIII and isolated again from agarose gel. This DNAfragment starts with an EcoRI sticky end followed by a BglII site andthe sequence from position—568 relative to the ATG codon up to the ATGcodon of the Saccharomyces cerevisiae phosphoglycerate kinase promoter.The ATG codon is followed by a BspMI site and a HindIII sticky end. Themulti-purpose cloning plasmid pTZ19R (obtained from Pharmacia) wascleaved with EcoRI and HindIII and ligated with the PGK promoterfragment, yielding pUR2918 (see FIG. 19). The plasmid was checked bymeans of sequence analysis.

Construction of plasmid PUR2920

For the fusion of the PGM promoter of pUR2918 to the xylanase gene thesynthetic DNA oligonucleotides shown in FIG. 20 (BAK14, 15, 18, 19, 20,21, 51, 52 and 53(SEQ ID NO:s 18,19,25,24,23,22,30, 31 and 32,respectively)) were annealed and ligated together resulting in thefragment BAK5. The oligonucleotides BAK51 and BAK53 (SEQ ID NO:s 30 and32, respectively) were not phosphorylated to prevent self-ligation ofthe resulting fragment, and the fragment was subsequently isolated fromagarose gel. The fragment BAK5 measures 169 bp and comprises theinvertase signal sequence and the mature xylanase gene up to the XhoIsite for a correct fusion to fragment BAK1 (SEQ ID NO:9) (see exampleIII). It differs from the earlier mentioned fragment BAK2 (SEQ ID NO:10)(example III) at both ends. At the 5′ end it contains a sticky end justbefore the second codon of the invertase signal sequence to obtain anexact fusion to the PGK promoter sequence in pUR2918. At the 3′ side ofthe XhoI site it contains an additional HindIII sticky end. The plasmidpUR2918 is cut with BspMI and HindIII and ligated to fragment BAK5 (SEQID NO:15) resulting in plasmid pUR2920 (see FIG. 21). The insertedfragment BAK5 was checked by means of sequence analysis. Plasmid pUR2920contains the Saccharomyces cerevisiae phosphoglycerate kinase (PGK)promoter, from nucleotide −568 relative to the ATG start codon, up tothe ATG start codon, the Saccharomyces cerevisiae invertase signalsequence correctly fused to this ATG codon, and the Aspergillus nigervar. awamori xylanase gene up to the SacI site. In order to simplify thecontinued construction the SacI site was changed to a XhoI site asdescribed in example III.

Construction of Plasmid pUR2922 and pUR2923

The 2 micron based episomal expression vector pUR2904 (see example III)was used to construct a plasmid vector for the expression of thexylanase gene regulated by the PGK promoter in S. cerevisiae. PlasmidpUR2920 was cleaved with BglII and XhoI and the 735 bp fragmentcontaining the PGK promoter, the invertase signal sequence and thexylanase gene up to the XhoI site was isolated from agarose gel. PlasmidpUR2904 was also cleaved with BglII and XhoI and the large vectorfragment was isolated. As a result of this digestion, the GAL7 promoterand invertase signal sequence were removed. This pUR2904 vector wasligated with the BglII-XhoI fragment of pUR2920, yielding pUR2922 (seeFIG. 22). Plasmid pUR2922 differs from the Saccharomyces cerevisiaeexpression vector pUR2904 (example III) as it contains the Saccharomycescerevisiae phosphoglycerate kinase promoter before the invertase signalsequence instead of the GAL7 promoter.

For the construction of a multi-copy integration vector with thePGK-xylanase expression cassette, plasmid pUR2792 served as startingpoint. Plasmid pUR2792 is a derivative of pMIRY2 (Lopes, 1989). Itcontains a BglII-HindIII polylinker instead of the BglII-HindIII partcontaining the S. oligorhiza DNA, and the part between the BalI site inthe pAT153 sequence and the HindIII site in the rDNA sequence has beendeleted. Plasmid pUR2792 was cleaved with BglII and HindIII and thevector band was isolated from agarose gel. The BglII-HindIII fragmentcontaining the PGK controlled xylanase expression cassette was isolatedfrom plasmid pUR2922 and ligated to pUR2792 vector that had been cleavedwith BglII-HindIII. The resulting plasmid pUR2923 (see FIG. 23) is aSaccharomyces cerevisiae multi-copy integration plasmid which containsthe Saccharomyces cerevisiae phosphoglycerate kinase promoter up to theATG start codon, the Saccharomyces cerevisiae invertase signal sequencefused to this promoter and the Aspergillus niger var. awamori maturexylanase gene fused in frame to the invertase signal sequence. Theintron (non-coding sequence) has been correctly removed from thexylanase gene.

Yeast cells of the Saccharomyces cerevisiae strain SU50 were transformedby the spheroplast method with plasmid pUR2923, linearized with HpaI(see example III). The resulting its+ transformed yeast cells wereanalyzed for xylanase production as described for the SU50 yeast cellswith the pUR2921 plasmid, with one alteration, the use of YPD medium (1%Yeast Extract, 2% Bacto peptone, 2% glucose) instead of YPG at the finalculturing stage. The expression level was about 10,000 units secreted in1 ml medium.

Production of Xylanase by PUR2923 Containing Yeast in Dough

Saccharomyces cerevisiae SU50 cells containing the pUR2923 plasmidmulti-copy integrated in the yeast chromosome were used in a baking testas described below. The increase in bread volume by the addition ofxylanase is caused by an enzymatic alteration of the starch tailings,through which the dough is capable of taking more advantage of thegassing activity of the yeast in the dough. A yeast with a high gassingpower is therefore required to obtain the full benefit of the additionof the xylanase enzyme. As the SU50 strain is a laboratory strain, itdoes not possess good gassing properties. For a baking experiment withthe xylanase producing SUSO yeast strain, supplementation with a goodgassing yeast strain is thus necessary.

The baking test described below was based on the 10 grams micro-loaftest (Shogren and Finney, 1984). The formulation of the dough was 10 gwheat flour (Columbus: MENEBA, The Netherlands); 0.15 g NaCl; 5.9 mlwater; 0.2 g pressed yeast (Koningsgist; Gist-Brocades, TheNetherlands). Supplementations to this formulation (xylanase producingand nonproducing yeast, xylanase enzyme) were dissolved in water justbefore mixing of the dough. Mixing took place for 5 minutes in a 10-grammixograph from National Manufacturing Co. Lincoln, Nebr. After mixing,the dough was fermented for 80 min. at 30° C. with two punches, onepunch at 40 min. and one at 80 min. Sheeting rolls used for the punchingwere spaced 2.0 mm. After fermentation the dough was moulded and proofedfor 70 din at 30° C. before baking. Baking took place for 12 min at 240°C. After weighing, the volume of the loaves was measured by means ofdwarf rapeseed displacement.

Supplementations to the dough were: Saccharomyces cerevisiae SU50 withpUR2923 (xylanase producing yeast), Saccharomyces cerevisiae SU50(parent strain) and purified xylanase enzyme. The SU50 yeast strainsused, were first grown on selective media: YNB w.o. amino acids (Difco)and 20 g/l glucose, supplemented with 60 mg/l leucine (SU50 parent only)and 20 mg/l histidine. These cultures were grown for 40 hours at 30° C.and then 5 ml was used to inoculate 45 ml of YPD (see above), and grownfor 16 hours at 30° C. Yeast cells were collected by centrifugation,washed once with fresh YPD, and centrifugated again. Various amounts ofthe (wet) pellet were resuspended in 5.9 ml of water just before mixingthe dough. When applied, the amount of purified xylanase added was 5 μlof a 40 U/μl solution (200 U). The effect of the varioussupplementations on the specific volume (S.V) of the bread is shown inthe table below:

Supplementation S.V. (ml/g) none 3.31 none 3.40  5 mg SU50 3.40 15 mgSU50 3.39 50 mg SU50 3.66  5 mg SU50; 200 U xylanase 3.78 15 mg SU50;200 U xylanase 3.97 50 mg SU50; 200 U xylanase 4.01  5 mg SU50:pUR29233.88 15 mg SU50:pUR2923 4.03 50 mg SU50:pUR2923 4.31

From the results shown in this table it is clear that the yeast strainproducing the xylanase (SU50:pUR2923) has a positive effect on thespecific volume of the bread, comparable to that of the addition ofpurified xylanase enzyme. The parent strain, when added in equivalentamounts, does not exhibit this effect. Of course this effect isaccomplished by blending of a bakers yeast with good gassing powercharacteristics, and an engineered laboratory yeast. The same positiveeffect, however, can be obtained when a bakers yeast with good gassingpower is engineered in a comparable way to produce the fungal xylanase.Furthermore, these yeast strains can be engineered to produce otherenzymes with bread improving capabilities (α-amylases, hemicellulasesetc.).

REFERENCES

Lopes, T. S., Klootwijk, J., Veenstra, A. E., van der Aar, P. C., vanHeerikhuizen, H., Raué, H. A. and Planta, R. J. (1989), Gene 79:199-206.

Shogren, M. D. and Finney, K. F. (1984), Cereal Chem. 61:418-423.

Tuite, M. F., Dobson, M. J., Roberts, N. A., King, R. M., Burke, D. C.,Kingsman, S. M. and Kingsman, A. J. (1982), EMBO Journal 1:603-608.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 43(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE:N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus niger var.awamori (B) STRAIN: CBS 115.52 (ATCC 11358) (vii) IMMEDIATE SOURCE: (B)CLONE: purified xylanase (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Ser AlaGly Ile Asn Tyr Val Gln Asn Tyr Asn Gly Asn Leu Gly Asp 1 5 10 15 Phe(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATESOURCE: (B) CLONE: oligo Xyl01 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:CCRTTRTART TYTGNACRTA RTT 23 (2) INFORMATION FOR SEQ ID NO:3: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE: oligo Xyl04 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:3: AAGTCGCCCA GGTTGCCGTT GTAGTTCTGGACGTAGTTGA TGCCGGC 47 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (vii) IMMEDIATE SOURCE: (B) CLONE: oligo Xyl05 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:4: AAGTCVCCNA RRTTVCCGTT GTA 23 (2) INFORMATIONFOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE:oligo Xyl06 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AAGTCSCCSAGGTTSCCGTT GTAGTTYTGS ACGTAGTTSA TSCCSGC 47 (2) INFORMATION FOR SEQ IDNO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE: primer Xyl11 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:6: GCATATGATT AAGCTGC 17 (2) INFORMATIONFOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 685 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A)ORGANISM: Aspergillus niger var. awamori (B) STRAIN: CBS 115.52 (ATCC11358) (vii) IMMEDIATE SOURCE: (B) CLONE: pAW14, pAW1 (ix) FEATURE: (A)NAME/KEY: intron (B) LOCATION: 231..279 (C) IDENTIFICATION METHOD:experimental (D) OTHER INFORMATION: /evidence= EXPERIMENTAL (ix)FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 1..81 (ix) FEATURE: (A)NAME/KEY: mat_peptide (B) LOCATION: join(82..230, 280..682) (C)IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /EC_number=3.2.1.8 /product= “endo-xylanase II” /evidence= EXPERIMENTAL /gene=“xylA” (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: join(1..230,280..685) (C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION:/EC_number= 3.2.1.8 /product= “pre-pro endo-xylanase II” /evidence=EXPERIMENTAL /gene= “xylA” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATGAAG GTC ACT GCG GCT TTT GCA GGT CTT TTG GTC ACG GCA TTC GCC 48 Met LysVal Thr Ala Ala Phe Ala Gly Leu Leu Val Thr Ala Phe Ala -27 -25 -20 -15GCT CCT GTG CCG GAA CCT GTT CTG GTG TCG CGA AGT GCT GGT ATT AAC 96 AlaPro Val Pro Glu Pro Val Leu Val Ser Arg Ser Ala Gly Ile Asn -10 -5 1 5TAC GTG CAA AAC TAC AAC GGC AAC CTT GGT GAT TTC ACC TAT GAC GAG 144 TyrVal Gln Asn Tyr Asn Gly Asn Leu Gly Asp Phe Thr Tyr Asp Glu 10 15 20 AGTGCC GGA ACA TTT TCC ATG TAC TGG GAA GAT GGA GTG AGC TCC GAC 192 Ser AlaGly Thr Phe Ser Met Tyr Trp Glu Asp Gly Val Ser Ser Asp 25 30 35 TTT GTCGTT GGT CTG GGC TGG ACC ACT GGT TCT TCT AA GTGAGTGACT 240 Phe Val ValGly Leu Gly Trp Thr Thr Gly Ser Ser Asn 40 45 50 GTATTCTTTA ACCAAAGTCTAGGATCTAAC GTTTTCTAG C GCT ATC ACC TAC TCT 295 Ala Ile Thr Tyr Ser 55GCC GAA TAC AGT GCT TCT GGC TCC TCT TCC TAC CTC GCT GTG TAC GGC 343 AlaGlu Tyr Ser Ala Ser Gly Ser Ser Ser Tyr Leu Ala Val Tyr Gly 60 65 70 TGGGTC AAC TAT CCT CAG GCT GAA TAC TAC ATC GTC GAG GAT TAC GGT 391 Trp ValAsn Tyr Pro Gln Ala Glu Tyr Tyr Ile Val Glu Asp Tyr Gly 75 80 85 GAT TACAAC CCT TGC AGC TCG GCC ACA AGC CTT GGT ACC GTG TAC TCT 439 Asp Tyr AsnPro Cys Ser Ser Ala Thr Ser Leu Gly Thr Val Tyr Ser 90 95 100 GAT GGAAGC ACC TAC CAA GTC TGC ACC GAC ACT CGA ACT AAC GAA CCG 487 Asp Gly SerThr Tyr Gln Val Cys Thr Asp Thr Arg Thr Asn Glu Pro 105 110 115 TCC ATCACG GGA ACA AGC ACG TTC ACG CAG TAC TTC TCC GTT CGA GAG 535 Ser Ile ThrGly Thr Ser Thr Phe Thr Gln Tyr Phe Ser Val Arg Glu 120 125 130 135 AGCACG CGC ACA TCT GGA ACG GTG ACT GTT GCC AAC CAT TTC AAC TTC 583 Ser ThrArg Thr Ser Gly Thr Val Thr Val Ala Asn His Phe Asn Phe 140 145 150 TGGGCG CAG CAT GGG TTC GGA AAT AGC GAC TTC AAT TAT CAG GTC ATG 631 Trp AlaGln His Gly Phe Gly Asn Ser Asp Phe Asn Tyr Gln Val Met 155 160 165 GCAGTG GAA GCA TGG AGC GGT GCT GGC AGC GCC AGT GTC ACG ATC TCC 679 Ala ValGlu Ala Trp Ser Gly Ala Gly Ser Ala Ser Val Thr Ile Ser 170 175 180 TCTTAA 685 Ser 185 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 211 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO:8: Met Lys Val Thr Ala Ala Phe Ala Gly Leu Leu Val Thr Ala PheAla -27 -25 -20 -15 Ala Pro Val Pro Glu Pro Val Leu Val Ser Arg Ser AlaGly Ile Asn -10 -5 1 5 Tyr Val Gln Asn Tyr Asn Gly Asn Leu Gly Asp PheThr Tyr Asp Glu 10 15 20 Ser Ala Gly Thr Phe Ser Met Tyr Trp Glu Asp GlyVal Ser Ser Asp 25 30 35 Phe Val Val Gly Leu Gly Trp Thr Thr Gly Ser SerAsn Ala Ile Thr 40 45 50 Tyr Ser Ala Glu Tyr Ser Ala Ser Gly Ser Ser SerTyr Leu Ala Val 55 60 65 Tyr Gly Trp Val Asn Tyr Pro Gln Ala Glu Tyr TyrIle Val Glu Asp 70 75 80 85 Tyr Gly Asp Tyr Asn Pro Cys Ser Ser Ala ThrSer Leu Gly Thr Val 90 95 100 Tyr Ser Asp Gly Ser Thr Tyr Gln Val CysThr Asp Thr Arg Thr Asn 105 110 115 Glu Pro Ser Ile Thr Gly Thr Ser ThrPhe Thr Gln Tyr Phe Ser Val 120 125 130 Arg Glu Ser Thr Arg Thr Ser GlyThr Val Thr Val Ala Asn His Phe 135 140 145 Asn Phe Trp Ala Gln His GlyPhe Gly Asn Ser Asp Phe Asn Tyr Gln 150 155 160 165 Val Met Ala Val GluAla Trp Ser Gly Ala Gly Ser Ala Ser Val Thr 170 175 180 Ile Ser Ser (2)INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:205 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATESOURCE: (B) CLONE: synthetic fragment BAK1 (xi) SEQUENCE DESCRIPTION:SEQ ID NO:9: GAATTCCTCG AGCGACTTTG TCGTTGGTCT GGGCTGGACC ACTGGTTCTTCTAACGCTAT 60 CACCTACTCT GCCGAATACA GTGCTTCTGG CTCCTCTTCC TACCTCGCTGTGTACGGCTG 120 GGTCAACTAT CCTCAGGCTG AATACTACAT CGTCGAGGAT TACGGTGATTACAACCCTTG 180 CAGCTCGGCC ACAAGCCTTG GTACC 205 (2) INFORMATION FOR SEQID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 202 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE:synthetic fragment BAK2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:GAATTCGAGC TCATCACACA AACAAACAAA ACAAAATGAT GCTTTTGCAA GCCTTCCTTT 60TCCTTTTGGC TGGTTTTGCA GCCAAAATAT CTGCGAGTGC TGGTATTAAC TACGTGCAAA 120ACTACAACGG CAACCTTGGT GATTTCACCT ATGACGAGAG TGCCGGAACA TTTTCCATGT 180ACTGGGAAGA TGGAGTCTCG AG 202 (2) INFORMATION FOR SEQ ID NO:11: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 113 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE: synthetic fragment BAK4(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GAATTCGCCG CGGGTATTAACTACGTGCAA AACTACAACG GCAACCTTGG TGATTTCACC 60 TATGACGAGA GTGCCGGAACATTTTCCATG TACTGGGAAG ATGGAGTCTC GAG 113 (2) INFORMATION FOR SEQ IDNO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 603 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE: PGK1promoter (PCR) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGAATTCAGATCTTGAATTG ATGTTACCCT CATAAAGCAC GTGGCCTCTT ATCGAGAAAG 60 AAATTACCGTCGCTCGTGAT TTGTTTGCAA AAAGAACAAA ACTGAAAAAA CCCAGACACG 120 CTCGACTTCCTGTCTTCCTA TTGATTGCAG CTTCCAATTT CGTCACACAA CAAGGTCCTA 180 GCGACGGCTCACAGGTTTTG TAACAAGCAA TCGAAGGTTC TGGAATGGCG GGAAAGGGTT 240 TAGTACCACATGCTATGATG CCCACTGTGA TCTCCAGAGC AAAGTTCGTT CGATCGTACT 300 GTTACTCTCTCTCTTTCAAA CAGAATTGTC CGAATCGTGT GACAACAACA GCCTGTTCTC 360 ACACACTCTTTTCTTCTAAC CAAGGGGGTG GTTTAGTTTA GTAGAACCTC GTGAAACTTA 420 CATTTACATATATATAAACT TGCATAAATT GGTCAATGCA AGAAATACAT ATTTGGTCTT 480 TTCTAATTCGTAGTTTTTCA AGTTCTTAGA TGCTTTCTTT TTCTCTTTTT TACAGATCAT 540 CAAGGAAGTAATTATCTACT TTTTACAACA AATATAAAAC AATGCGCAGC AGGTAAGCTT 600 GGG 603 (2)INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATESOURCE: (B) CLONE: primer PGP01 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:GGAATTCAGA TCTTGAATTG ATGTTACCCT CATAAAGCAC GTG 43 (2) INFORMATION FORSEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE:primer PGP02 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCCAAGCTTACCTGCTGCGC ATTGTTTTAT ATTTGTTGTA AAAAGTAGAT AATTACTTCC 60 (2)INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:208 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATESOURCE: (B) CLONE: synthetic fragment BAK5 (xi) SEQUENCE DESCRIPTION:SEQ ID NO:15: GAATTCGAGC TCATCACACA AACAAACAAA ACAAAATGAT GCTTTTGCAAGCCTTCCTTT 60 TCCTTTTGGC TGGTTTTGCA GCCAAAATAT CTGCGAGTGC TGGTATTAACTACGTGCAAA 120 ACTACAACGG CAACCTTGGT GATTTCACCT ATGACGAGAG TGCCGGAACATTTTCCATGT 180 ACTGGGAAGA TGGAGTCTCG AGAAGCTT 208 (2) INFORMATION FORSEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:AATTCGAGCT CATCACACAA ACAAACAAAA CAAAATGATG CTTTTGCAAG CC 52 (2)INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO:17: TTCCTTTTCC TTTTGGCTGG TTTTGCAGCC AAAATA 36 (2) INFORMATION FORSEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:TCTGCGAGTG CTGGTATTAA CTACGTGCAA AACTAC 36 (2) INFORMATION FOR SEQ IDNO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: AACGGCAACCTTGGTGATTT CACCTATGAC GAGAGT 36 (2) INFORMATION FOR SEQ ID NO:20: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GCCGGAACAT TTTCCATGTACTGGGAAGAT GGAGTC 36 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:21: CATTTTGTTT TGTTTGTTTG TGTGATGAGC TCG33 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO:22: AGCCAAAAGG AAAAGGAAGG CTTGCAAAAG CAT 33 (2) INFORMATIONFOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:AATACCAGCA CTCGCAGATA TTTTGGCTGC AAAACC 36 (2) INFORMATION FOR SEQ IDNO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ACCAAGGTTGCCGTTGTAGT TTTGCACGTA GTT 33 (2) INFORMATION FOR SEQ ID NO:25: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GGAAAATGTT CCGGCACTCTCGTCATAGGT GAAATC 36 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:26: TCGAGACTCC ATCTTCCCAG TACAT 25 (2)INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO:27: AATTCGCCGC GGGTATTAAC TACGTGCAAA ACTA 34 (2) INFORMATION FORSEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:ACCAAGGTTG CCGTTGTAGT TTTGCACGTA GTTAATACCC GCGGCG 46 (2) INFORMATIONFOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:TCGAGACTCC ATCTTCCCAG TACAT 25 (2) INFORMATION FOR SEQ ID NO:30: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: AATGATGCTT TTGCAAGCCTTCCTTTTCCT TTTGGCTGGT TTTGCAGCCA AAATA 55 (2) INFORMATION FOR SEQ IDNO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GCCGGAACATTTTCCATGTA CTGGGAAGAT GGAGTCTCGA GA 42 (2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: AGCTTCTCGAGACTCCATCT TCCCAGTACA T 31 (2) INFORMATION FOR SEQ ID NO:33: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: AATTCCTCGA GCGACTTTGTCGTTGGTCTG GGCTGGACCA CTGGTTCT 48 (2) INFORMATION FOR SEQ ID NO:34: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: TCTAACGCTA TCACCTACTCTGCCGAATAC AGT 33 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:35: GCTTCTGGCT CCTCTTCCTA CCTCGCTGTGTACGGCTGG 39 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:36: GTCAACTATC CTCAGGCTGA ATACTACATCGTCGAGGATT AC 42 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:37: GGTGATTACA ACCCTTGCAG CTCGGCCACAAGCCTTGGTA C 41 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:38: CAGACCAACG ACAAAGTCGC TCGAGG 26 (2)INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO:39: GTAGGTGATA GCGTTAGAAG AACCAGTGGT CCAGCC 36 (2) INFORMATION FORSEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:GTAGGAAGAG GAGCCAGAAG CACTGTATTC GGCAGA 36 (2) INFORMATION FOR SEQ IDNO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: TTCAGCCTGAGGATAGTTGA CCCAGCCGTA CACAGCGAG 39 (2) INFORMATION FOR SEQ ID NO:42: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GTTGTAATCA CCGTAATCCTCGACGATGTA GTA 33 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:43: CAAGGCTTGT GGCCGAGCTG CAAGG 25

What is claimed is:
 1. A purified recombinant DNA material comprising anucleotide sequence encoding a ripening from of a β-1,4 endoxylanase offungal origin having bread improving activity, said nucleotide sequencebeing selected from the group consisting of the nucleotide sequence ofSEQ ID NO:7, a nucleotide sequence encoding the amino acid sequenceencoded by SEQ ID NO:7, a nucleotide sequence which hybridizes to thenucleotide sequence of SEQ ID NO:7, said hybridization being performedin 6×SSC at 68° C. followed by wash steps in 2×SSc and 0.4×SSC,respectively at 68° C., a nucleotide sequence containing SEQ ID NO:7which encodes a mature form of β-1,4 endoxylanase, a nucleotide sequencecontaining SEQ ID NO:7 which encodes a preform of β-1,4 endoxylanase, anucleotide sequence containing SEQ ID NO:7 which encodes a proform ofβ-1,4 endoxylanase and a nucleotide sequence containing SEQ ID NO:7which encodes a preproform of β-1,4 endoxylanse.
 2. The recombinant DNAmaterial according to claim 1, wherein said nucleotide sequence encodinga ripening form of a β-1,4 endoxylanase is of Aspergillus origin.
 3. Therecombinant DNA material according to claim 1, wherein said nucleotidesequence encoding a ripening form of a β-1,4 endoxylanase is ofAspergillus niger origin.
 4. The recombinant DNA material according toclaim 1, wherein said nucleotide sequence encoding a ripening form of aβ-1,4 endoxylanase is a Aspergillus niger var. awamori origin.
 5. Therecombinant DNA material according to claim 1, wherein said nucleotidesequence encoding a ripening form of β-1,4 endoxylanase encodes SEQ IDNO:8.
 6. The recombinant DNA material according to claim 1 comprisingDNA with a nucleotide sequence encoding the mature form of β-1,4endoxylanase.
 7. The recombinant DNA material according to claim 1,further encoding at least one other enzyme having amylolytic orhemicellulolytic of cellololytic activity.
 8. A transformed host cellcomprising recombinant DNA material according to claim 1, such that saidhost cell expresses and secretes the ripening form of β-1,4 endoxylanaseencoded by said recombinant DNA material.
 9. The transformed host cellaccording to claim 8, wherein said host cell is selected from the groupconsisting of a bacterial cell, a fungal cell, a yeast cell and a plantcell.
 10. The transformed host cell according to claim 9, wherein saidhost cell is a fungal cell selected from the genera Aspergillus andTrichoderma.
 11. The transformed host cell according to claim 9, whereinsaid host cell is a fungal cell selected from the species Aspergillusniger var. awamori, Aspergillus niger var. niger, Aspergillus nidulansand Aspergillus oryzae.
 12. The transformed host cell according to claim9, wherein said host cell is a bacterial cell selected from the generaBacillus, Lactobacillus and Streptococcus, or a yeast cell of the generaSaccharomyces, Kluyveromyces, Hansenula and Pichia.
 13. The transformedhost cell according to claim 9, wherein said host cell is selected froma yeast cell of species Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Kluyveromyces lactis, Kluyveromyces marxianus, Hansenulapolymorpha and Pichia pastoris.
 14. A purified form of a β-1,4endoxylanase encoded by the recombinant DNA material according toclaim
 1. 15. A process for producing a ripening form of a β-1,4endoxylanase, said ripening form comprising SEQ ID NO:8, said processcomprising: culturing a transformed host cell according to claim 8 in anutrient medium such that said endoxylanase is produced, and optionallyisolating said endoxylanase.
 16. A bread improver composition comprisingthe β-1,4 endoxylanase according to claim
 14. 17. A bread improver,flour or dough composition comprising a cell according to claim
 8. 18. Abread improver, flour or dough composition comprising a cell accordingto claim
 9. 19. A bread improver, flour or dough composition comprisinga cell according to claim
 10. 20. A flour or dough compositioncomprising a ripening form of β-1,4 endoxylanase according to claim 14.21. A bakery product comprising the composition according to claim 20.22. A process for preparing a bakery product by baking a flourcomposition wherein the improvement is adding the composition accordingto claim
 20. 23. A method of processing a cellulose-containing rawmaterial to prepare beer, paper, starch, or gluten, or to decomposecellulose- or hemicellulose-containing waste which comprises contactingsaid raw material with a ripening form of the β-1,4-endoxylanaseaccording to claim
 14. 24. The method of claim 23, wherein the ripeningform of β-1,4 endoxylanase which is used is a mature form.
 25. Themethod of claim 23, wherein the ripening form of β-1,4 endoxylanasewhich is used is of Aspergillus origin.
 26. The method of claim 23wherein the cellulose-containing raw material is agricultural wastewhich is contacted with said β-1,4 endoxylanase to produce animal feed.27. The method of claim 23 wherein said cellulose-containing rawmaterial is waste from paper mills.
 28. A method of processing acellulose-containing material to prepare beer, paper, starch or glutenor to decompose cellulose- or hemi-cellulose containing waste whichcomprises contacting said material with the transformed host cellaccording to claim 8 such that said cell secretes a ripening form of theβ-1,4 endoxylanase in situ.
 29. The method of claim 28 wherein the saidripening form of β-1,4 endoxylanase is a mature form.
 30. The method ofclaim 28 wherein said ripening form of β-1,4 endoxylanase is ofAspergillus origin.
 31. The method of claim 28 wherein saidcellulose-containing material is agricultural waste which is contactedwith the β-1,4 endoxylanase to produce animal feed.
 32. The method ofclaim 28 wherein said cellulose-containing material is waste from papermills.
 33. A recombinant DNA material comprising a nucleotide sequenceencoding a ripening form of a polypeptide having β-1,4 endoxylanaseactivity wherein said nucleotide sequence is selected from the groupconsisting of SEQ ID NO:7 and a nucleotide sequence encoding thepolypeptide encoded by SEQ ID NO:7.
 34. A purified form of β-1,4endoxylanase encoded by the recombinant DNA material according to claim33.
 35. An isolated polypeptide comprising SEQ ID NO:8.
 36. Therecombinant DNA material according to claim 33, further encoding atleast one other enzyme having amylolytic, hemicellulolytic orcellulolytic activity.
 37. A transformed host cell comprising therecombinant DNA material of claim
 33. 38. A bread improver, flour ordough composition comprising at least one of the purified forms of β-1,4endoxylanase of claim 34 or a transformed host cell comprising arecombinant material comprising a nucleotide sequence encoding aripening form of a polypeptide having β-1,4 endoxylanase activitywherein said nucleotide sequence is selected from the group consistingof SEQ ID NO:7 and a nucleotide sequence encoding the polypeptideencoded by SEQ ID NO:7.
 39. A bakery product comprising the compositionaccording to claim
 38. 40. The method of processing acellulose-containing raw material to prepare beer, paper, starch, orgluten, or to decompose cellulose- or hemicellulose-containing wastewhich comprises contacting said raw material with a ripening form of theβ-1,4 endoxylanase according to claim
 34. 41. A method of processing acellulose-containing material to prepare beer, paper, starch or glutenor to decompose cellulose- or hemi-cellulose containing waste whichcomprises contacting said material with the transformed host cellaccording to claim 37 such that said cell secretes a ripening form ofthe β-1,4 endoxylanase in situ.